Labor unrest is a closely watched issue in Spain after its biggest union said on Friday that a general strike over planned government austerity measures was likely.
Workers for state-owned rail network Renfe have already struck over working conditions in February and March.
Saturday's call made no reference to a strike called for June 8 by civil servants, who are angry over pay cuts imposed by government austerity measures.
In a statement, the Federal Railway Union (SFF) said it would stage rallies and call for further strikes in June if Renfe's management failed to negotiate employment guarantees and other working conditions.
After protests in Greece turned violent in early May, there are concerns Spain could follow the same route, as the government enforces drastic spending cuts in an effort to convince markets its public finances are under control.
Economists have said a general strike would be unlikely to draw wide participation, even from unionized workers, since many of them hold the relatively safe permanent contracts and could be wary of jeopardizing them by walking out.