The Russian gas company Gazprom has stopped transiting gas to Hungary via Ukraine, according to Kyiv.
Ukrainian industry officials claim that gas is now bypassing the country to Budapest through a Black Sea pipeline.
A new contract was agreed by Hungary and Russia this week to transport 4.5 billion cubic metres of natural gas for 15 years, starting on Friday.
The pipeline will flow south of Ukraine through the Black Sea, Austria, and Serbia before reaching Hungary.
But the deal has angered Kyiv as Ukraine will lose its status as the main transit country for Russian natural gas to the European Union, as well as billions of euros in revenue.
"For decades, Hungary has received gas via the territory of Ukraine, and the Ukrainian side has not even violated its obligations," the head of the Ukrainian gas transport company, Sergiy Makogon, said on Friday.
"The strengthening of the dominant position of one player and their use of leverage for obviously political purposes against the backdrop of a shocking rise in gas prices in Europe must be stopped."
Ukraine's foreign ministry had earlier issued a statement saying that Hungary's supply deal was a "purely political, economically unreasonable decision".
But Hungarian Prime Minister Viktor Orban has dismissed the criticism in an interview on public radio.
Kyiv has called on the United States and Germany to impose sanctions on Gazprom over the new deal
"A joint statement from the United States and Germany said that if the Kremlin used gas as a weapon, there would be an appropriate response," Yuriy Vitrenko, the head of Ukraine's Naftogaz said on Facebook.
"We are now waiting for the imposition of sanctions on a 100% subsidiary of Gazprom, the operator of Nord Stream 2."
Ukraine has long accused Russia of deliberately using natural gas as a weapon against Ukraine, an allegation which Moscow has rejected.
Kremlin spokesman Dmitry Peskov told reporters that Russia was continuing to fulfill its existing obligations and denied violating any existing treaty.
"There have been and will be accusations against Russia, the majority of which are politicised," Peskov added.
Relations between the two countries have been tense since Russia annexed the Ukrainian Black Sea peninsula of Crimea in 2014.