A convoy of 262 Russian trucks was expected at the Ukrainian border on Wednesday as Kiev vowed to block the aid mission from its territory over fears it was a ploy to bolster pro-Kremlin rebels.
The convoy of what Russia says is humanitarian aid has sparked fears of an escalation in a conflict that has left hundreds dead in eastern Ukraine in recent months and plunged Moscow's ties with the West to their lowest point in decades.
The United States backed Ukraine's demand for border checks to clear up concerns that Moscow is seeking to support rebels.
Washington and its European allies have been voicing concern for days over the convoy, which sources told Russian news agencies would arrive at the border on Wednesday.
"Russia has no right to move into Ukraine unilaterally, whether under the guise of humanitarian convoys or any other pretext, without Kiev's permission," State Department spokeswoman Marie Harf told reporters.
Ukrainian officials "have a plan in place that they feel comfortable with. We feel comfortable with it as well. And now the Russians need to deliver," Harf said.
France warned earlier that Russia could use the operation as "a cover" for sending in troops, echoing concerns from Berlin and London.
Kiev said the trucks would be stopped at the border for any aid to be unloaded and transported into conflict-torn eastern Ukraine with the help of the International Committee of the Red Cross.
"We will not allow (the aid) to be accompanied by the Russian ministry for emergency situations or by Russian troops," said Valeriy Chalyy, deputy head of the presidential administration.
However Moscow was adamant the convoy would reach its destination, calling for "maximum cooperation" from Ukraine to ensure the aid was delivered to the besieged rebel strongholds of Lugansk and Donetsk.
The three-kilometre-long (1.9-mile) aid convoy left Moscow on Tuesday carrying 2,000 tonnes of "humanitarian supplies", including medical equipment, baby food and sleeping bags, Russian media reported.
As fierce fighting continued in the industrial east, Ukraine's military said six servicemen had been killed and 31 were wounded in the past 24 hours.
Seven civilians were also injured in shelling overnight in Donetsk, local authorities said, while Ukraine's military said it was ready to surround the rebels' second city of Lugansk.
Kiev's forces hope to cut off rebel access to the porous Russian border, where NATO says Moscow has massed 20,000 troops.
Fighting between government forces and pro-Moscow rebels also pushed Ukraine's currency, the hryvnia, to a historic low, trading at 13.14 against the dollar, although the central bank insisted this was only a "temporary trend".
Confusion over aid mission
Russia has been pressing for a humanitarian mission to the east, where four months of fierce battles have left cities without power, running water or fuel, and with dwindling food supplies.
President Vladimir Putin said Monday that Moscow was sending aid in response to the "catastrophic consequences" of Kiev's offensive against insurgents.
Kiev has said it will only accept aid as part of a broader international mission involving Europe and the US under the supervision of the Red Cross.
Moscow -- which denies allegations it is seeking to boost the insurgents who are losing ground to Ukraine's military -- has insisted it is working with the Red Cross and that the convoy would not include military personnel.
But, adding to the confusion, the Red Cross said no green light had been given for an aid mission. "We still need to get some more information before we can move ahead," ICRC spokeswoman Anastasia Isyuk told AFP in Geneva.
Russia has also said the route of the convoy had been agreed with Kiev.
The Russian foreign ministry said the lorries would cross the border near the Russian town of Shebekino, into government-controlled territory, but there were fears the convoy could take a different route farther east into Ukraine, across a rebel-held stretch of the border.
"Russia expects maximum cooperation by the Ukrainian side in ensuring the security of the delivery of humanitarian aid," the foreign ministry said in a statement.
French President Francois Hollande said he had "grave concerns" about the mission, which his Foreign Minister Laurent Fabius warned "could be a cover by the Russians to set themselves up near Donetsk and Lugansk and declare a fait accompli".
Meanwhile in Moscow, police detained dozens of protesters who had gathered outside the Ukrainian embassy to show their support for Kiev.
The private Moscow Echo radio station said about 200 people gathered for the demonstration before being dispersed by the police.
"The police were fairly rough with the women who were dressed in the colours of the Ukrainian flag," a Moscow Echo reporter said.
Over 1,300 people have been killed in four months of what the Red Cross has already deemed a civil war in Ukraine, while 285,000 have fled their homes, according to the United Nations.