Here we report that PLC-β1 catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-β1 mutational analysis revealed the importance of His331 and His378 for the catalysis. The expression of PLC-β1 and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-β1 activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-β1 is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC-β1 catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-β1 that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC-β1 and that c-Jun binding activity is significantly increased by insulin and PLC-β1 overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-β1. These results hint at the fact that PLC-β1 catalytic activity signals a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.