Deltex4 (DTX4) is a member of the Deltex family of proteins. To date several lines of evidences suggest that Deltex family of proteins is closely linked to cell development and cell differentiation. However, little is known about the role of DTX4 in adipogenic differentiation. In this study, we assessed the impact of DTX4 on adipogenic differentiation in vitro, we found that DTX4 protein expression gradually increased during adipogenic differentiation of 3T3-L1 preadipocytes cell line. While DTX4 stable knockdown by recombinant shRNA lentivirus (sh-DTX4) notably reduced the number of lipid droplets and down-regulated the expression of adipogenic transcription factors C/EBPα and PPARγ and adipogenic markers gene FABP4 and Adipsin. Besides, cell numbers and incorporation of 5-Ethynyl-2'-deoxyuridine (EdU) into cells were significantly decreased during mitotic clonal expansion (MCE) in sh-DTX4 cells postinduction. Furthermore, compared to recombinant shRNA lentivirus control group (sh-CON), the mRNA levels of Wnt signaling genes such as Wnt6, Wnt10b and β-catenin, were obviously elevated in sh-DTX4 group at day 3 of postinduction. Taken together, our results indicate that DTX4 stable knockdown inhibits adipogenesis of 3T3-L1 cells through inhibiting C/EBPα and PPARγ, arresting mitotic clonal expansion and regulating Wnt signaling pathway.