Pre-mRNA splicing is a complex and dynamic process in which protein phosphorylation and dephosphorylation both play important roles. Although specific phosphatases, such as PP1 and PP2A, have been implicated in splicing, direct evidence for their involvement has been lacking, and their exact function(s) in this process remain unknown. In this study, we show that PP1 and certain PP2A family phosphatases play essential but redundant roles in splicing. Unexpectedly, we found that these phosphatases are required principally for the second step of the splicing reaction. Furthermore, we provide evidence that components of U2 and U5 snRNPs, specifically SAP155 and U5-116 kDa, are the key spliceosomal substrates for these phosphatases. Based on these data, we propose that dephosphorylation of U2 and U5 snRNP components by PP1/PP2A family phosphatases facilitates essential structural rearrangements in the spliceosome during the transition from the first to the second step.