The gathering was held alongside an official summit of South American presidents, including Hugo Chavez, the president of Venezuela and a close Morales ally.
Venezuela would "not stand idle" in the face of any action aimed at destabilising Morales' government, Chavez said.
"Here we are, with you, resisting the aggression, because we feel it is our responsibility," Chavez told the crowd of 40,000 in a stadium in Cochabamba.
Following sometimes violent pro-autonomy demonstrations in the wealthy region of Santa Cruz this week, local leaders have renewed their demands for more regional independence from the national government in La Paz. They have called a public meeting for Friday at which they will decide their next step.
Leaders of the regions of Pando, Tarija and Beni are organising similar meetings next week.
Opposition to some of Morales' policies, such as his plan to redistribute idle farmland to peasant farmers, is strongest in those regions.
Both Morales and Chavez have accused the US of conspiring with the opposition.
"The struggle continues against North American imperialism ... the people together with the armed forces and its government will never allow them to boycott this process of change," Morales said.