The row will provide ammunition to Brexit campaigners, who argue that European law is tying the hands of government ministers on a range of policy areas.
The Commission has come under fire after it issued a letter of formal notice - the first stage in infringement proceedings - requesting further explanation of the HGV levy from the UK authorities.
The HGV levy was introduced in April 2014 for vehicles weighing 12 tonnes or more. The aim, says the UK, is to ensure these vehicles make a contribution to the wear and tear of the road network.
RELATED CONTENT
Levy amounts vary according to the vehicle's weight, axle configuration, and journey duration.
UK-registered vehicles pay levy costs at the same time and in the same transactions as vehicle excise duty (VED). Vehicles registered abroad must make levy payments before entering the UK. The levy can be paid by day, week, month, or year.
However, national users benefit from a reduction of vehicle taxes, meaning that national users are de facto not paying the road charge.
The Commission says, "Within the EU, it is the prerogative of member states to introduce road charges. If a member state wishes to make foreign users pay for the use of national roads, that charge must apply to all users."
But it adds, "After thorough analysis, however, we are concerned that the levy discriminates against non-UK hauliers."
The UK has two months to respond to the Commission and should the executive consider the reply unsatisfactory, it will consider sending a reasoned opinion to the UK.
Germany is considering a similar toll scheme for private cars and it also faces possible EU action after passing a law that ensures that only German-registered benefit from a deduction of its road charge.
UK transport minister Patrick McLoughlin described the levy as a massive boost to British hauliers under pressure from foreign rivals.
It was designed, he said, to compensate for levies and tolls that British lorries pay when travelling overseas that do not exist in Britain.
David Campbell-Bannerman, a Conservative MEP, criticised the threat of possible action.
He said, "The charge allows us to recover some of the costs of foreign lorries using UK roads for free. It is time foreign lorries - and the whole EU - paid their own way," he said. "It is yet another example of our nation's total loss of control."
UKIP Transport spokesman Jill Seymour MEP said,
"The EU does not own our roads and they should mind their own business"
"This is another example of how undemocratic the EU Commission can be - it should only ever be up to the UK government to decide what taxes we charge foreign vehicles."
"The Commission clearly has no respect for our sovereignty"
"This will just give voters in the UK even more of a reason to vote leave on June 23."
On Thursday, a UK government spokesperson told this website: "Our levy is justified and consistent with the free movement of goods. British drivers regularly pay when they use the roads in other countries, and it is fair that foreign HGV drivers should do the same here.
"The Commission has written to us seeking our views about the levy and we will respond in due course."
The Road Haulage Association has also strongly criticised the Commission's decision to challenge the legality of the HGV road user levy.
Commenting, RHA chief executive Richard Burnett said: "This challenge is nonsense. The levy breaks neither the letter nor spirit of EU law. Brussels was fully briefed during its design and implementation - and continental hauliers continue to pay the levy without complaint.
"This is an important and successful measure that addresses an issue of real concern; to both the haulage industry and wider public."
He added, "Foreign operators - who now account for almost 90 per cent of international trade - paid nothing at all to use our roads and the full burden of contributing towards road maintenance fell on UK-registered trucks. The levy changed that, within a framework designed by the EU itself."