IGF-I enhances steroidogenesis in granulosa cells by stimulating the expression of the rate-limiting steroidogenic enzyme, cytochrome P-450 side-chain cleavage (P-450_scc). This effect is mediated through an IGF response element (IGFRE) that binds polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF) and Sp1. Sp1 is essential for activation of the IGFRE, and PSF functions as a repressor. We investigated mechanisms of modulation of the IGFRE by the atypical protein kinase C (PKC)ι in a porcine stable granulosa cell line, JC-410. PKCι was found in nuclear extracts, and levels were increased by IGF-I after 24 and 48 h of treatment. Immunoprecipitation experiments demonstrated that PSF and PKCι associated with each other in nuclear extracts from JC-410 cells. Transient transfection with expression plasmids of kinase-active and kinase-deficient PKCι isoforms enhanced transcriptional activity of the IGFRE regardless of kinase catalytic activity. Depletion of PKCι protein by small interfering RNA suppressed basal IGFRE activity but did not prevent IGF-I stimulation of the IGFRE. We conclude that PKCι enhances transcriptional activity of the porcine P-450_scc IGFRE independently of kinase activity by a mechanism involving protein-protein interaction with PSF.