Abstract: Objective To explore the effects of vitamin C and niacinamide on the growth and differentiation of human primary cultured keratinocytes. Methods Normal human foreskin was used in this study. The epidermis was separated enzymatically from the dermis by thermolysin, and keratinocytes were isolated from the epidermis by digestion with trypsin plus EDTA. The single keratinocytes were cultured with underlying NIH-3T3 cells as feeder cells in a complete medium supplied with 50 mg/L （vitamin C group）, niacinamide of 400 μmol/L （niacinamide group） or vehicle （control group）. Immunocytochemistry and immunodot blot were performed using monoclonal antibodies directed against C-myc, cyclin D1, filaggrin and involucrin. Results The colony number was highest in vitamin C group, followed by the control group and niacinamide group, and the colony morphology in vitamin C group was similar to that in the control group, but distinct from that in the niacinamide group. A significant increase was noticed in the expression of C-myc, cyclin D1, filaggrin and involucrin in vitamin C-treated keratinocytes compared with the control keratinocytes （all P < 0.05）; however, in niacinamide-treated keratinocytes, the expression of filaggrin was significantly enhanced （P < 0.01）, that of involucrin remained unchanged （P > 0.05）, while that of C-myc was depressed （P < 0.05）. Conclusions These results demonstrate that vitamin C has a favorable effect on both the growth and differentiation of human keratinocytes, while niacinamide seems to only promote the differentiation but attenuate the growth of human keratinocytes.