Keratinocyte migration is a key aspect of re-epithelialization during wound healing. Matric metalloproteinase 9 (MMP9) contributes to this process and deificiencies in the MMP9 lead to impaired healing. Inappropriate expression of MMP9 also contributes to impaired re-epithelialization. Previously we demonstrated that FOXO1 was activated in wound healing but to higher levels in diabetic wounds. To address mechanisms of impaired re-epithelialization we examined MMP0 expression in vivo in full thickness dermal scalp wounds creared in experimental K14.Cre+.Foxo1L/L mice with lineage-specific Cre recombinase deletion of floxed FOXO1 and compared the results to control littermates. MMP9 was induced during wound healing but at a significantly higher level in diabetic compared to normal wounds. FOXO1 deletion substantially blocked this increase. By chromatin immunoprecipitation FOXO1 was shown to bind to the MMP9 promoter, FOXO1 overexpression increased MMP9 transcriptional activity and increased MMP9 expression simulated by high glucose that was blocked by FOXO1 deletion or FOXO1 knockdown. We also show for the first time that high glucose impairs keratinocyte migration by inducing high levels of MMP9 expression in diabetic wound healing, which represents a novel mechanism for impaired re-epithelialization in diabetic wounds.