Steroids are synthesized in adrenal glands and gonads under the control of pituitary peptides. These peptides bind to cell surface receptors to activate the cyclic AMP (cAMP) signaling pathway leading to an increase of steroidogenic gene expression. Exactly how cAMP activates steroidogenic gene expression is not clear, except for the knowledge that transcription factor SF-1 plays a key role. Investigating the factors participating in SF-1 action, we found that c-Jun and homeodomain-interacting protein kinase 3 (HIPK3) were required for basal and cAMP-stimulated expression of one major steroidogenic gene, CYP11A1. HIPK3 enhanced SF-1 activity, and c-Jun was required for the functional interaction of HIPK3 with SF-1. Furthermore, after cAMP stimulation, both c-Jun and Jun N-terminal kinase (JNK) were phosphorylated through HIPK3. These phosphorylations were important for SF-1 activity and CYP11A1 expression. Thus, we have defined HIPK3-mediated JNK activity and c-Jun phosphorylation as important events that increase SF-1 activity for CYP11A1 transcription in response to cAMP. This finding has linked three common factors, HIPK3, JNK, and c-Jun, to the cAMP signaling pathway leading to increased steroidogenic gene expression.