The exact mechanism by which ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling is not known. ENPP1 contains two somatomedin-B-like domains (i.e. SMB 1 and 2) involved in ENPP1 dimerization in animal cells. The aim of the present study was to investigate if these domains modulate ENPP1 inhibitory activity on insulin signaling in human insulin target cells (HepG2). ENPP1 (ENPP1-3'myc), ENPP1 deleted of SMB 1 (ENPP1-ΔI-3'myc) or of SMB 2 (ENPP1-ΔII-3'myc) domain were cloned in frame with myc tag in mammalian expression vector pRK5. Plasmids were transiently transfected in human liver HepG2 cells. ENPP1 inhibitory activity on insulin signaling, dimerization and protein-protein interaction with insulin receptor (IR), reported to mediate the modulation of ENPP1 inhibitory activity, were studied. As compared to untransfected cells, a progressive increase of ENPP1 inhibitory activity on insulin-induced IR β-subunit autophosphorylation and on Akt-S(473) phosphorylation was observed in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. Under non reducing conditions a 260 kDa homodimer, indicating ENPP1 dimerization, was observed. The ratio of non reduced (260 kDa) to reduced (130 kDa) ENPP1 was significantly decreased by two thirds in ENPP1-ΔII-3'myc vs. ENPP1-3'myc but not in ENPP1-ΔI-3'myc. A similar ENPP1/IR interaction was detectable by co-immunoprecipitation in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. In conclusion, SMB 1 and SMB 2 are negative modulators of ENPP1 inhibitory activity on insulin signaling. For SMB 2 such effect might be mediated by a positive role on protein dimerization.