Connective tissue growth factor (CTGF) plays an important role in the proliferation of hepatic progenitors, however, little is known concerning the mechanism(s) through which it influences their differentiation. The differentiation of hepatic progenitors (WB-F344), either stimulated with recombinant CTGF or stably transfected with a CTGF overexpression plasmid, was investigated. Expression of the differentiation markers α-fetoprotein (AFP), albumin (ALB) and cytokeratin-19 (CK-19) was assessed. To confirm the effects of CTGF on progenitor differentiation, cells were treated with an inhibitor (WP631) of CTGF. Treatment of WB-F344 cells with recombinant CTGF for 24 h did not change the survival rate significantly, but the progenitors were enlarged with a decreased nuclear/cytoplasmic ratio. CTGF downregulated the expression of the fetal hepatocyte marker, AFP, while it upregulated the mature hepatocyte cell marker, ALB. The effect of CTGF overexpression plasmid on WB-F344 cell differentiation was consistent with a pattern of direct CTGF stimulation, including decreased AFP and increased ALB expression. Furthermore, the suppression of CTGF induction by an inhibitor was associated with significant inhibition of hepatic progenitor cell differentiation into hepatocytes. Importantly, we showed that differentiated WB-F344 cells by CTGF had in vitro functions characteristic of hepatocytes, including ALB production, glycogen storage and cytochrome P450 activity. Both recombinant CTGF and the CTGF overexpression plasmid induced hepatic progenitor differentiation into hepatocytes. This was suppressed by the CTGF inhibitor.