Objective: To provide a new and simple strategy for keratinocyte transplantation. Methods: Keratinocyte suspension and dermal substitutes including acellular dermal matrix (ADM) and collagen membrane were prepared. Full-thickness skin defect wounds of Balb/c-nu mice were created and the mice were assigned into 5 groups: the control group, where the wound was grafted with keratinocyte suspension only; the one-step ADM and collagen membrane groups, where the wound was grafted with ADM or collagen membrane plus dripping of keratinocyte suspension; the two-step ADM and collagen membrane groups, where keratinocyte suspension was dripped 2 weeks after ADM or collagen membrane grafting. The percentage of epithelium was observed. Results: Two weeks after grafting, no evidence of re-epithelialization was seen in the one-step ADM group, and the re-epithelialization rate in the one-step collagen membrane group, two-step ADM group and two-step collagen membrane group was 54.1, 41.8 and 76.8%, respectively, indicating that the re-epithelialization rate in the two-step collagen membrane group was higher than that in the other two groups (p < 0.01). Conclusions: Keratinocyte suspension combined with dermal substitutes grafting offers a more flexible way of making use of keratinocytes. Compared with ADM, microporous collagen membrane is a more preferable option for combined use with keratinocyte suspension for skin grafting.