Besides using for hair removal, depilatory agents have been considered to be used as a penetration enhancer for transepidermal drug delivery. To examine the effect in hair follicles (HFs), two commercially available depilatory creams were tested on the dorsal skin of mice to monitor the effect deep into the skin structure. Fifteen male BALB/c mice were used in this study. Depilatory creams were applied to the dorsal skin of the same animal using shaved and untouched treatments as controls to minimize individual differences. Skin samples were collected at three days, one week and two weeks (n = 5 for each) after the treatment, and subjected for hematoxylin-eosin staining, and immunohistochemical analysis for proinflammatory cytokines. The morphological examination showed an increase in the thickness of epidermal layer of the depilatory cream-treated skin at early time points and in the subcutis at two weeks. Depilatory cream promoted entry of anagen phase and increased the number of hair follicles in the subcutis at one and two weeks. Immunohistochemistry showed elevated percentages of dermal fibroblasts expressing interleukin-6, tumor necrosis factor-α, and tumor necrosis factor-β. Shaving process increased the thickness of epidermis and dermis as depilatory creams did, but did neither induce the expression of proinflammatory cytokines in the dermal fibroblasts nor the number of HFs. The results suggested that the commercially available depilatory creams caused a transient minor inflammatory response of the skin and increased the levels of cytokines that might subsequently affect hair growth.