Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.