Users of progestin-only contraceptives have raised protein S (PS) levels compared with baseline. This contrasts with the reduction in PS levels observed in users of combined oral contraceptives, which contain both a progestin and an estrogen. To determine the effect of progesterone and other progestin isoforms on the expression of PS and to describe the mechanism involved. Promoter activity of the PROS1 gene that encodes PS was assessed in vitro using breast and liver carcinoma cell lines grown in the presence of various progestins, with and without the addition of excess progesterone receptors. An electromobility shift assay (EMSA) was also performed to identify the progesterone receptor binding element. PROS1 transcriptional levels were directly upregulated by 25% by progesterone via a mechanism that was progesterone receptor isoform B (PR-B)-dependent. The process was blocked by the progesterone receptor modulator RU486. Results for the EMSA demonstrated that a probe comprising nucleotides -397 to -417 of the PROS1 promoter bound to ligand-activated PR-B, suggesting that the domain is a progesterone response element (PRE). The type of progestin isoform greatly influenced the level of PROS1 promoter upregulation, with medroxyprogesterone able to stimulate a > 2-fold stronger response compared with progesterone. The PROS1 promoter is responsive to progesterone and other progestins via a mechanism involving PR-B interacting with a PRE. The type of progestin is important as some elicit stronger upregulatory effects than others, which may influence the choice of progestin used for hormonal contraception by PS-deficient individuals.