Dressed in his army general's uniform, Castro saluted thousands of people as they passed the reviewing stand at the Plaza of the Revolution in the Cuban capital.
As the guest of honor, Maduro, the leader of Cuba's closest ally and longtime benefactor, was at his side.
The annual rally and parade is being held this year against a backdrop of a thaw in Cuban relations with the US and a deepening crisis in oil-rich Venezuela, which has undercut public support for Maduro.
"We will only advance toward the normalization of bilateral relations on a basis of respect for the sovereignty, the independence of Cuba, which includes the lifting of the blockade and the return of territory usurped by the US naval base at Guantanamo," Guilarte said.
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Negotiations on restoring relations have been underway since Castro and US President Barack Obama's surprise announcement December 17 that they would work to normalize ties severed for more than 50 years.
Cuba's opening to the United States fits with a broader effort to overhaul the island's Soviet-style economy and attract foreign investment needed to spur growth.
Since the collapse of the Soviet Union, Cuba has relied heavily on Venezuela for cut-rate oil supplies and investment, a lifeline that could be threatened by its ally's economic problems.
But Guilarte, saluting Maduro's presence at the May Day rally, warned: "We firmly reject all external meddling, as well as destabilizing actions, violence and war that violate constitutional order, sovereignty, independence and the self-determination of the Venezuelan people."
More than 2,000 members of unions and political groups from around the world also were invited to attend.
Guilarte said the idea was to send to the world "a genuine message of unity, commitment and majority support of the Cuban people for the revolution, for Fidel and for Raul.