Mastercard is being investigated by the [http://vienne.co/tui-xach vienne] over fees charged for card transactions made by people visiting Europe.
The Commission said some of the firm's inter-bank fees and related practices may be anti-competitive.
The commission is already investigating rival Visa over similar practices.
Mastercard, which said it would fully co-operate with regulators, could be fined up to $740m, or 10% of its 2012 revenue, if found guilty.
The credit card firm said that it always aimed to balance the interests of both consumers and retailers.
'Crucial importance'The investigation will examine payments made by people from outside the European Economic Authority (EEA) - the EU's 27-member states as well as Norway, Liechtenstein and Iceland - who use their credit and debit cards when inside the area.
The EU said the main aim of its probe was to ensure consumers were not harmed.
When a US tourist uses a Mastercard to make a purchase in [the European Economic Area], these fees can be quite high, generally much higher than those paid in Europe, European Commission spokesman Antoine Colombani said.
In 2007, a similar probe led to the Commission banning Mastercard from charging cross-border fees within the European Economic Area.
The Commission said payment cards were of crucial importance for cross-border and internet payments.
It is therefore a priority for the European Commission to prevent competition distortions in inter-bank arrangements on fees, it said.
As well as inter-bank fees paid by cardholders from non-EEA countries, the Commission said the probe would look at rules that obliged merchants to accept all types of Mastercard cards, even if some of them incur higher charges.
The probe will also look at Mastercard's restrictions on merchants who wish to use banks outside their own country, which could be cheaper.
Ultimately, such behaviour is liable to slow down cross-border business and harm EU consumers, it said.
The Commission said it would submit proposed regulation by the middle of the year on inter-bank fees aimed at ensuring a level playing field for all card providers.