The spliceosome catalyzes nuclear pre-mRNA splicing via formation of an intron lariat and is arguably the most complex macromolecular machine in eukaryotic cells. Intron lariat formation is a conservative feature of the splicing reaction for both spliceosomal and group II introns. Despite the importance of the lariat formation in pre-mRNA splicing, an atomic-level understanding of the reaction mechanism remains elusive. In this work, a quantum mechanics and molecular mechanics method with thermodynamic integration is used to calculate the free-energy profile along the reaction pathway. The results demonstrate that the catalytic Mg2+ ion does not act as a Lewis acid to activate the nucleophile and the intron lariat is formed via a dissociative transition state stabilized by electrostatic interactions with two catalytic Mg2+ ions. Proton transfer occurs from the nucleophile to the leaving oxygen group through the phosphate substrate after the transition state is reached, which is important for stabilization of the intron-lariat product and the efficiency of pre-mRNA splicing. The two-metal-ion mechanism proposed in this study provides a novel insight into understanding of the splicing reaction catalyzed by the spliceosome.