The influence of sodium and lithium content in the metasilicate crystal structure (SiO32−) is herein analyzed concerning the heterogeneous transesterification reaction for biodiesel production. Na2SiO3 and Li2SiO3 were characterized structurally and microstructurally. The catalyst content was evaluated between 1 and 5 wt %, where the maximum conversions to FAME (∼99%) were obtained using 3 wt % of Na2SiO3 during 1 h at 65 °C. After some cyclic experiments, it was determined that Na2SiO3 possesses a better stability and consequently reutilization capacity than Li2SiO3 in terms of its triglycerides conversion to FAME. DFT calculations were then used to analyze these experimental differences, revealing significant differences between these two catalysts in terms of energy, geometrical configuration, and electronic structure. It was found that three active sites are required on both catalytic surfaces to overcome the methanol deprotonation, which is herein suggested as the rate-controlling step of the entire transesterification mechanism. In one site, the oxygen atom of methanol approaches, while Lewis acid and Brønsted base sites are needed for the methoxide anion stabilization and proton stabilization, respectively.