"This a special day in the history of the EU and of Croatia," European Parliament president Martin Schulz said as he received the new members who were elected in April.
The lawmakers earlier took part in a ceremony outside the parliament building attended by Schulz, European Commission chief Jose Manuel Barroso and Croatian speaker Josip Leko.
Zagreb's accession to the EU is "a signal of hope for the future of a united Europe," Schulz said.
A Croatian navy choir sang the European anthem in Croatian.
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With the arrival of the Croatian lawmakers, the European Parliament now has 766 members working in 24 languages.
After the 2014 European elections, the number will be reduced to a total of 751 in line with the Lisbon Treaty.
The European Commission from today will also have one new member -- Neven Mimica, a former deputy prime minister of Croatia who is the new commissioner for consumer protection.