The involvement of the apoptosis signal-regulating kinase 1 (ASK1)-related signalling pathway in the control of reproduction is unknown. This study aimed to investigate the role of ASK-1 in the control of basic ovarian functions (proliferation, apoptosis and hormone release) and its response to ovarian hormonal regulators (leptin and FSH). We compared the accumulation of ASK-1, proliferation marker proliferating cell nuclear antigen (PCNA), apoptosis marker Bax and apoptosis and proliferation regulating transcription factor p53 and the release of progesterone (P4), oxytocin (OT), insulin-like growth factor I (IGF-I) and prostaglandins F (PGF) and E (PGE) using cultured porcine ovarian granulosa cells transfected with ASK-1 cDNA and cultured with leptin or FSH. This study is the first to demonstrate that ASK-1 does not affect cell apoptosis and viability in ovarian cells, but promotes cell proliferation, suppresses p53, alters the release of ovarian hormones (P4, OT, IGF-I, PGF and PGE) and defines their response to the upstream hormonal regulators leptin and FSH. Therefore, ASK-1 can be considered a new and important regulator of multiple ovarian functions.