We have shown previously that prostratin, a non-tumor promoting phorbol ester, inhibits chikungunya virus (CHIKV)-induced cytopathic effects in vitro. Prostratin is a potent activator of protein kinases C (PKC), a family of related serine/threonine kinases that regulate many cellular processes such as proliferation and apoptosis. The objective of this study was to explore the mechanism of the anti-CHIKV activity of prostratin. Prostratin reduced the production of infectious virus particles and viral protein accumulation in a dose-dependent manner at a post-entry step during virus replication. The antiviral effect of the compound was cell-dependent, with potent antiviral activity observed in human skin fibroblasts cells, the primary target cells of CHIKV infection. The antiviral activity of prostratin was markedly reduced in the presence of PKC inhibitors, therefore confirming that the antiviral effect results from an activation of PKCs. Together these results showed that PKCs are potential host targets for the inhibition of CHIKV replication.