Abstract: Oral wounds exhibit advantageous healing characterized by rapid wound closure with less scar formation. Wound inducible transcript-3.0 (wit3.0) has been identified as a differentially expressed cDNA in the oral wound. The goal of this study was to complete the characterization of wit3.0 gene and peptide structures and to delineate the function of wit3.0 during wound healing. The in silico analyses revealed that the wit3.0 gene structure was highly conserved among mammalian species. The deduced peptide sequence contained two predicted coiled-coil domains that were homologous to a portion of myosin heavy chain II coiled-coil domain. Wit3.0 immunostaining in wounded gingival fibroblasts revealed its association with the actin cytoskeleton, and Western blot suggested that wit3.0 could potentially form a peptide complex in fibroblasts. From these data, we have postulated that wit3.0 may participate in fibroblast contraction. Unlike other pro-contractile peptides such as α-SM actin influenced by TGF-beta1, the expression of wit3.0 appeared to be regulated by a constitutive mechanism specific to oral fibroblasts. In a series of in vitro studies, we found that over-expressing wit3.0 in oral as well as skin fibroblasts accelerated the collagen gel contraction; while suppressing the wit3.0 gene expression through siRNA treatments decreased the gel contraction. Two non-synonymous single nucleotide polymorphisms (SNPs) have been reported in the wit3.0 gene. The SNP-derived amino acid substitution resulted in a stronger contraction profile in vitro. Although the allelic frequencies of less than 1%, the wit3.0 SNP genotype could affect wound healing processes. For an in vivo model using mouse full-thickness excisional wound, we developed a gene delivery method using lentiviral vector incorporated with cationized gelatin. When wit3.0-expression vector was transduced in mouse skin wounds, the early wound closure was significantly accelerated. Together, these data suggest that wit3.0 is a new member of the cytoskeleton-associated proteins, which plays a role in the fibroblast-driven tissue contraction. The increased wit3.0 expression appears to facilitate the advantageous wound closure as seen in oral wounds. The outcome of this study may provide the basis for a potential treatment modality aiming to accelerate the skin wound closure, and warrant the future study of wit3.0 in wound healing.