The EU's diplomatic mission at the Geneva-based WTO said it had filed a formal request for consultations with Russia, the first step towards a full-blown trade dispute.
In Brussels' sights are the anti-dumping duties that Russia has slapped on so-called "light commercial vehicles" (LCVs) made by Germany and Italy, both of which are leading global players in the auto sector.
"The EU believes the anti-dumping duties are incompatible with WTO law, both on procedural and on substantive grounds," the EU mission said in a statement.
Moscow rejected EU claims of any wrongdoing.
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"We consider that our measure fulfils our WTO obligations completely. We're ready to work closely with a panel to find a solution to this dispute," Maxim Medvedkov, Russia's chief trade negotiator, was quoted as saying by the news agency Ria Novosti.
WTO members are allowed to impose extra duties when goods are being "dumped" on them or sold at below market prices to grab business.
The EU argues that "the duties of 23 per cent to 29.6 per cent imposed on European LCVs are significantly hampering access to the Russian market."
In 2012, the year Russia joined the WTO, EU LCV exports to the country were worth more than 100 million euros (USD 137 million), it said.
Exports have been declining since Russia imposed a "recycling fee" on cars, trucks, buses and other vehicles, days after joining the WTO, it added.
"The anti-dumping measures subject of today's panel request are further choking off EU exports of LCVs," the EU said.
Brussels and Moscow are also wrangling at the WTO over embargoes on a string of goods from ex-communist countries, several of which are now members of the 28-nation EU.