"It can only deepen the crisis in the region, which Russia itself has created and has continued to fuel," he said in the statement.
"This is a blatant breach of Russia's international commitments... And a further violation of Ukraine's sovereignty by Russia," he added.
Rasmussen poured doubt on Moscow's claim that the convoy, which had been waiting at the border for a week, was actually meant for humanitarian purposes.
"The disregard of international humanitarian principles raises further questions about whether the true purpose of the aid convoy is to support civilians or to resupply armed separatists," he said.
The transatlantic military alliance accuses Moscow of actively providing weapons and fighters to pro-Kremlin separatists as well as massing its own troops near the border.
The latest developments "are even more worrying as they coincide with a major escalation in Russian military involvement in eastern Ukraine since mid-August, including the use of Russian forces," Rasmussen said.
"Instead of de-escalating the situation, Russia continues to escalate it, despite the efforts of the international community to find a political solution to the crisis," he said.
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