The human ovarian surface epithelium (hOSE) is a squamous-to-cuboidal layer that surrounds the ovary. hOSE undergoes injury and repair cycles as a result of ovulation-induced inflammation, an event relevant to the development of epithelial ovarian cancer (EOC). Locally produced steroids mediate the response to inflammation. 3β-Hydroxysteroid dehydrogenase (3β-HSD) drives the intracrine generation of progestogens and androgens that potentially affect cell survival and proliferation. We therefore investigated the regulation of 3β-HSD along with downstream steroid signalling in hOSE. Double immunofluorescence of cultured primary hOSE cells confirmed the expression of 3β-HSD protein Interleukin (IL). IL-1α treatment of primary cells to mimic ovulation-associated inflammation suppressed 3β-HSD1 expression and stimulated 3β-HSD2 mRNA (P < 0.001), without affecting total 3β-HSD protein and activity or androgen or progesterone receptor (PR) mRNA levels. Conversely, IL-4 as a proxy for a post-ovulatory healing cytokine increased both 3β-HSD transcripts, total protein and activity (P < 0.01). IL-4 also suppressed androgen receptor expression (P < 0.01) without affecting that of the PR, thereby potentially sustaining both progesterone biosynthesis and its underlying signalling in the ovarian surface. 3β-HSD protein was immunodetectable in primary ascites of women who were diagnosed with EOC but both mRNA transcripts were diminished relative to normal cells (P < 0.05). Notably, this difference was countered by IL-4 treatment (P < 0.01). We conclude that stimulation by IL-4 could be physiologically relevant to post-ovulatory ovarian healing and suggest a novel therapeutic strategy for the activation of progesterone-associated apoptosis in ovarian cancer. Also, our results suggest an attenuation of 3β-HSD expression in EOC although further studies are required for confirmation.