The vessel was greeted with fireworks and cheers from a crowd that had gathered at the Cocoli locks to celebrate.
Panama’s President Juan Carlos Varela described the waterway as a route that would unite the world.
Juan Carlos Varela thanked the nearly 30,000 people who had worked on the canal’s expansion.
Construction on the new lane for Panama Canal, which runs for 48 miles, began in 2007 and was due to finish in 2014.
However, strikes and disputes over costs delayed the $5.2 billion project.
The original Panama Canal was first used in August 1914. It was built by the US and handed over to local control in 1999.
The expansion allows a new, much-larger generation of container ships to pass through the isthmus.
Some 35 to 40 vessels transit the waterway everyday.
Panama hopes the expansion will increase the revenues it gets from the canal, reported to have been $2.6 billion in 2015.
However, the canal could face competition from a new passage in Nicaragua.
The controversial 172 mile scheme, being built by a Chinese company, will be longer, deeper and wider than the Panama Canal.
Panama Canal’s construction started in 2014 and it is estimated to cost $50 billion.
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