The start of the lock expansion was celebrated at a ceremony jointly presided over by Panama President Ricardo Martinelli, Italian Prime Minister Silvio Berlusconi and Spanish Deputy Premier Manuel Chaves.
The function was also attended by the presidents of Colombia, Guatemala and Honduras.
The project is being carried out by Grupo Unidos por el Canal, a consortium led by Spain's Sacyr Vallehermoso construction company.
The canal, designed in 1904 for ships with an 876-foot length and 92-foot width, is too small to handle the post-Panamax ships that are three times as big, making it necessary to expand by building the new set of locks.
The $5.25 billion expansion project will build a third set of locks that will be able to handle container ships with maximum capacity of 12,000 TEUs, compared to the current 4,500 TEUs.
The new locks will be 1,400 feet long and 180 feet wide and will accommodate ships with drafts of up to 50 feet. The existing locks are 1,000 feet long and 110 feet wide.