The spliceosome (SPL) is a majestic macromolecular machinery composed of five small nuclear RNAs and hundreds of proteins. SPL removes noncoding introns from precursor messenger RNAs (pre-mRNAs) and ligates coding exons, giving rise to functional mRNAs. Building on the first SPL structure solved at near-atomic-level resolution, here we elucidate the functional dynamics of the intron lariat spliceosome (ILS) complex through multi-microsecond-long molecular-dynamics simulations of ∼1,000,000 atoms models. The ILS essential dynamics unveils ( i ) the leading role of the Spp42 protein, which heads the gene maturation by tuning the motions of distinct SPL components, and ( ii ) the critical participation of the Cwf19 protein in displacing the intron lariat/U2 branch helix. These findings provide unprecedented details on the SPL functional dynamics, thus contributing to move a step forward toward a thorough understanding of eukaryotic pre-mRNA splicing.