Spliceosome dephosphorylation is essential control step that enables intron removal from pre-mRNA for the regulation of gene expression. However, the phosphatase that is biologically responsible has not been identified. Here we show that PP2A B́ η, a B subunit of PP2A phosphatase, associates with spliceosome B* to C* complex and dephosphorylates spliceosome regulators by the most-conserved its binding motif that can be absolutely regimented interaction with PP2A substrates.