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The Pope is the first pontiff to visit the Communist Caribbean island.
Speaking in the capital, Havana, he said freedom of conscience was "the basis and foundation of all other human rights".
"A modern state cannot make atheism or religion one of its political ordinances," he added.
The Cuban President, Fidel Castro, who was educated in a Jesuit school, declared Cuba an atheist state after he took power in 1959.
In 1992 Cuba dropped its commitment to atheism from its constitution and last December Christmas was reinstated as an official holiday ahead of the Pope's five-day visit.
Today the Pope urged an end to Cuba's political and economic isolation: "She needs to open herself to the world and the world needs to draw close to Cuba".
He also condemned the "resurgence of a certain capitalist neo-liberalism which subordinates the human person to blind market forces" and "often places unbearable burdens upon less favoured countries.
"We thus see a small number of countries growing exceedingly rich at the cost of increasing impoverishment of a great number of countries," he said.
President Castro, whom the Church blames for decades of persecution of Catholics, urged every able-bodied Cuban to attend the papal mass, if not out of conviction, at least out of respect.
And according to BBC correspondent Tim Gibb there was a mood of intense excitement in a crowd of several hundred thousand.
During the address in Havana's Revolution Square, which lasted more than three hours, the Pope was constantly interrupted by people chanting, "Long live Juan Pablo; he wants everyone to be free!"
The president, sitting in the front row, listened intently and smiled occasionally while alongside him sat Nobel-winning novelist Gabriel Garcia Marquez of Colombia.
The Mass, like his earlier one in the eastern city of Santiago de Cuba, was also broadcast on television nationwide.
The Pope looked tired at times during the long ceremony, but he was not too weary to joke about it. "I am not against applause, it allows the Pope to rest," he said on one occasion with a wry smile.
This morning he called for the release of Cuban political prisoners at a Mass on the outskirts of Havana at one of the country's principal shrines.
There are an estimated 500 prisoners of conscience in Cuba. Opposition activists face constant harassment and arrest.
Earlier in the week, the Pope personally handed Fidel Castro a list of names which the Church would like to see released.
So far there's been no public response from the Cuban government; however the country's vice-president, Carlos Lahe said the list would be given proper attention.
In his farewell speech the following day, the Pope condemned the economic embargo imposed on Cuba by the United States, describing it as an indiscriminate measure that hurt the poor.
On his return, the Pope described the trip as unforgettable, and a great event of spiritual, cultural and social reconciliation for the island.
He compared the five-day Cuban trip to the one he made to his native Poland in 1979 and hoped it would set off a train of events that would benefit the Cuban people.
Two weeks later his prayers were answered when Cuba released more than 300 political prisoners as an act of goodwill.
In March 1998, the US announced it was ready to allow the return of direct flights to Cuba to increase the flow of humanitarian aid to the Cuban people.
But it was not until October 2000 that the US House of Representatives approved the sale of food and medicines to Cuba. In November 2001 the US exported food to Cuba for the first time in more than 40 years after a request from the Cuban government to help it cope with the aftermath of Hurricane Michelle.
But tensions between Cuba, the US and Europe resurfaced in 2003 after a crackdown on dissidents that saw the execution of three men who tried to escape to America.
In May 2004 new US sanctions restricted US-Cuba family visits and cash remittances from expatriates. President Castro led a protest march against measures.
The Pope died on 2 April 2005 after he failed to recover from a throat operation.
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