Steroid sulphatase (STS) is the primary enzyme for desulphating steroids from their inactive to their active forms. Principal substrates include steroid precursors oestrone-sulphate and dehydroepiandrosterone sulfate. Alterations in STS activity can directly affect local concentrations of oestradiol, testosterone and dihydrotestosterone; steroids that are frequently dysregulated in disease. Despite the importance of STS activity on steroid synthesis, little is known about its regulation. In vitro studies on breast and prostate cancer have indicated that TNFα may in part regulate STS activity. To investigate how inflammation regulates STS, we examined TNFα effects in vitro and in vivo. Colorectal cancer (CRC) cells were treated with TNFα and STS activity measured using 3HE1S desulphation. In vivo, STS activity was measured by the same method in multiple tissues of TNF-Tg mice, which globally overexpress TNFα, and compared findings to age matched WT control animals. Tissues analysed included liver, heart, kidney, spleen, lung, bowel, skin and omental fat. Consistent with other findings, TNFα treatment increased STS activity in CRC cells, suggesting this response occurs in multiple cancer types. The glycosylation inhibitor tunicamycin significantly (P<0.01) inhibited TNFα-induced STS activity rise, suggesting a post-translational modification is important for activity. STS activity was generally increased in most organs of TNF-Tg mice when compared to WT controls, with significant rises seen in liver (2.73.6 nmol/mg per h, P=0.0052), spleen (406715 pmol/mg per h, P=0.006) and large bowel (312365 pmol/mg per h P=0.0002). Here we demonstrate both in vitro and in vivo that TNFα regulates STS activity in multiple tissue types. This increase in STS activity is not just a phenomenon seen in malignancy, but instead may be a normal physiological response to inflammation. In the acute setting this could be a homeostatic mechanism, however the effects of chronic increases in STS activity remains unknown.