It has been shown recently that triterpenes inhibit cancer cell growth of various cell types in vitro. In this work, the effect of highly purified triterpenes (TE) with betulin as the major compound (>80% w/w) on cell proliferation, apoptosis, and differentiation of human keratinocytes was analyzed in vitro, ex vivo, and in vivo. In vitro, TE increased calcium influx into primary keratinocytes and upregulated various differentiation markers including keratin 10. TE also specifically increased the expression of the non-selective transient receptor potential canonical (subtype) 6 (TRPC6) in keratinocytes, and knocking down TRPC6 inhibited keratin 10 upregulation. Ex vivo, in human skin explants TE induced the expression of TRPC6 in the epidermis and increased DNA fragmentation of terminally differentiating keratinocytes. Topical treatment with TE of actinic keratoses, that represent in situ squamous cell carcinomas with disturbed epithelial differentiation, resulted in downgrading of aberrant Ki67 expression and upregulation of keratin 10 in vivo. Our data indicate that TE promotes keratinocyte differentiation in vitro and in vivo. This effect seems to be mediated at least in part by TRPC6.