Protein phosphatase 1 (PP1) and Ca²⁺/calmodulin-dependent protein kinase (CaMKII) are up-regulated in heart disorders. Alterative splicing factor (ASF), a major splice factor for CaMKII splicing, can be regulated by both protein kinase and phosphatase. Here we determine the role of PP1 isoforms in ASF-mediated splicing of CaMKII in cells. We found that (1) PP1, but not α or β isoform, enhanced the splicing of CaMKII in HEK293T cells; (2) PP1 promoted the function of ASF, evidenced by the existence of ASF-PP1 association as well as the PP1 overexpression- or silencing-mediated change in CaMKII splicing in ASF-transfected HEK293T cells; (3) CaMKII splicing was promoted by overexpression of PP1 and impaired by application of PP1 inhibitor 1 (I1PP1) or pharmacological inhibitor tautomycetin in primary cardiomyocytes; (4) CaMKII splicing and enhancement of ASF-PP1 association induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) were potentiated by overexpression of PP1 and suppressed by inhibition of PP1 with I1PP1 or tautomycetin in primary cardiomyocytes; (5) Functionally, overexpression and inhibition of PP1 respectively potentiated or suppressed the apoptosis and Bax/Bcl-2 ratio, which were associated with the enhanced activity of CaMKII in OGD/R-stimulated cardiomyocytes; (6) CaMKII was required for the OGD/R induced- and PP1 exacerbated-apoptosis of cardiomyocytes, evidenced by a specific inhibitor of CaMKII KN93, but not its structural analog KN92, attenuating the apoptosis and Bax/Bcl-2 ratio in OGD/R and PP1-treated cells. In conclusion, our results show that PP1 promotes the alternative splicing of CaMKII through its interacting with ASF, exacerbating OGD/R-triggered apoptosis in primary cardiomyocytes.