A convoy of 280 Russian trucks reportedly packed with aid headed for eastern Ukraine on Tuesday, but Ukraine said it would not let the mission in because it is not being coordinated by the International Red Cross and could be a covert military operation.
The neutral agency said it had no information on what the trucks were carrying or where they were going.
That has raised fears in Ukraine and the West, where leaders have voiced concerns that Russia could use the initiative as a pretext for sending troops into separatist-held territory.
The goods would be stopped at the border and transferred to other vehicles, Reuters news agency reported, citing Ukrainian presidential aide Valery Chaly.
The trucks, laden with food and medical supplies departed Naro-Fominsk on Tuesday after an agreement was initially reached between Russia and Ukraine on Monday to allow a Red Cross-led humanitarian mission into the eastern region of Luhansk. Luhansk has borne the brunt of the fighting, and food and energy supplies are running short.
Al Jazeera's Rory Challands, reporting from Moscow, said that once across the border, the much-needed supplies would be distributed by Ukrainian authorities and that there were other pre-conditions to the delivery.
Our correspondent said Ukraine insisted it would be an internationally coordinated effort and that the convoy must cross at a point controlled by the military, not the pro-Russia separatists.
"It has to go to a border that is controlled by the Ukrainian army and Ukraine essentially has to be the party that takes it across the border and distributes it in the country," our correspondent said.
The convoy will be accompanied by representatives of the Organization for Security and Cooperation in Europe (OSCE) once it is inside Ukraine, a mediator said.
The Russian cargo as well as aid from the United States and the European Union will be delivered under the auspices of the International Red Cross and escorted by OSCE representatives through north-eastern Ukraine, former Ukrainian President Leonid Kuchma told Interfax news agency.
NATO has previously expressed concerns that Russia could use a humanitarian mission to launch an invasion of Ukraine's east, which is slowly being reclaimed from pro-Russian separatists by the Ukrainian military.
"Russia says this is all fully compliant with international laws on delivering aid, but lots of people are very twitchy about this, including the International Red Cross who say they will allow no armed escorts for humanitarian convoys," our correspondent said.
"NATO has been saying for days that the chances of a Russian invasion are rising and a humanitarian convoy could be used as cover for this."
Russia has told reporters the trucks will be taking 400 tons of cereals, 100 tons of sugar, 62 tons of baby food, 54 tons of medical drugs and stock, as well as 12,000 sleeping bags and 69 power generators to the civilians of Luhansk.
Russia initially came up with a proposal to dispatch an international humanitarian mission to the region last Tuesday at the UN Security Council.
The Interfax-Ukraine news agency on Monday quoted Ukrainian Foreign Ministry spokesman Yevhen Perebiynis as saying the humanitarian convoy could only be carried out under specific conditions.
At least 100km of the Russian-Ukrainian border is currently in rebel hands.