Most of the crude glycerol produced globally is generated by biodiesel production, which makes this byproduct an environmental responsibility of the biofuel industries. Among the forms of this compound in use, microalgae cultivation is a promising alternative that may generate a reduction in crude glycerol treatment costs via using it as an organic, carbon-rich substrate in culture media. In this work, the influence of different concentrations of glycerol in the culture medium, the composition of fatty acids and proteins in Spirulina sp. LEB 18 biomass and their effect on its growth were investigated. The fatty acid profile of the biomass was altered, showing a 20% increase in the unsaturated concentration and a 60% reduction in the saturated concentration in the culture supplemented with 0.05 mol L-1 of glycerol compared to those in the control. The addition of the substrate stimulated an increase in its cellular concentration (3.00 g L-1, 0.05 mol L-1), productivity (0.72 g L-1 d(-1), 0.05 mol L-1) and its protein production (69.78% w w(-1), 0.05 mol L-1).