Rajoy, speaking Sunday at youth leadership conference in Madrid, said he will not allow "anyone to play games with national sovereignty" in Spain and would not engage in "games of division, fighting and a return to the past," the Spanish news agency EFE reported.
The Spanish premier made the remarks as pro-independence sentiment has surged in Spain's largest and most economically powerful region, marked by a rally in September during which hundreds of thousands of Catalans formed a 250-mile human chain to call for an independent state.
Catalan President Artur Mas of the center-right Convergence and Union has pledged to hold a referendum on the region's political future, perhaps as early as next year.
In the meantime, direct negotiations are being held with Madrid over a new constitutional arrangement between Catalonia and Spain as relations between the two sides have deteriorated.
Madrid has flatly refused to consider the possibility of a independence referendum even as the pro-secession movement has gained steam, fueled by feelings Catalonia's financial and social interests are being brushed aside.
Rajoy used his speech at the National Convention of New Generations to rally support for national cohesion, asserting Spain is "a great nation," Europe's oldest and longest-living.
Spain "is bound by ties of all kinds -- emotional, personal, historical, and by its future and progress," he said, emphasizing that the Spanish Constitution guarantees the rights and freedoms of all Spaniards and has generated the most decentralized state model of the history of the country.
This, he said, has led to the largest increase in living standards in many centuries.
"We have to be proud of who we are -- Spanish -- because we have a great country and a great nation," Rajoy declared. "We will write the future of Spain for all the Spanish, no one will be left out of the decisions that affect us all, and I will not accept anyone playing games with national sovereignty, because that is what I think and because I'm the president the government."
The lines brought applause and chants of "Viva Spain!" and "I am Spanish!" by the young audience, EFE reported.
Rajoy insisted that there are still many things for Spain and Catalonia to do together, such as working within the European Union, carrying out more integration and removing borders and tariffs to create "a better world."
"What we will not accept ... are the games of division, confrontation and return to the past. We are in the future, and the future is Spain and Europe."
The prime minister's speech came on the same day the Catalan National Assembly at an extraordinary weekend meeting in Barcelona overwhelmingly rejected a push to stage an independence referendum independent of negotiations with Madrid.
In a victory for Pere Navarro, first minister of the Catalan Socialist Party, the council voted 258-41 to oppose a push by some members of the regional Parliament to transfer to power to hold a referendum from the executive to the legislative body, El Pais reported.