Heparanase is a heparan sulphate endo-glycosidase which was previously detected in the outer root sheath of murine hair follicles. Heparanase overexpression was reported to improve mouse hair (re)growth. In this study, we investigated its involvement in human hair biology. Immunofluorescence detection was used to explore heparanase distribution in both anagen and catagen hair follicles. Heparanase functionality was assessed in in vitro cultured hair follicles, in the presence of a heparanase activity inhibitor. Our results showed that heparanase expression was (i) primarily located in the inner root sheath (IRS) of human hair follicle, and there (ii) restricted to anagen phase. Furthermore, inhibition of heparanase in in vitro cultured hair follicles induced a catagen-like process. Hair shaft retreat upward was accompanied by a decrease in Ki67-positive cells, the formation of an epithelial strand as evidenced by K14 keratin expression, and the loss of IRS as assessed by transglutaminase 1 and desmoglein labelling. IRS distribution of heparanase and the induction of catagen-like involution of hair follicles when a potent heparanase inhibitor is added suggest that heparanase is a key actor of IRS differentiation and hair homeostasis.