GH and PRL stimulate proliferation and insulin production of pancreatic β-cells. Whereas GH- and PRL-regulated transcription of the insulin gene in insulinoma cells has been shown to depend on STAT5 (signal transducer and activator of transcription 5), the signaling pathways involved in GH/PRL-induced β-cell replication are unknown. The roles of various signaling pathways in human GH (hGH)-induced DNA synthesis were studied by analysis of the effect of specific inhibitors in both the insulin-producing cell line, INS-1, and in primary β-cells. The mitogen-activated protein kinase kinase (MEK)-inhibitor, PD98059, as well as the mitogen-activated protein kinase p38 (MAPKp38) inhibitor, SB203580, partially inhibited hGH- induced proliferation in INS-1 cells but had no significant effect in primary β-cells. Staurosporine, a protein kinase C (PKC) and protein kinase A (PKA) inhibitor, blocked both basal and hGH-induced proliferation in INS-1 cells, but had no inhibitory effect in primaryβ -cells. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited hGH-induced proliferation neither in INS-1 cells nor in primary β-cells, whereas the tyrosine kinase inhibitor, genistein, completely inhibited hGH- induced proliferation in both primary β-cells and INS-1 cells. To analyze the possible role of STAT5 in hGH-induced proliferation, a dominant negative STAT5 mutant, STAT5Δ749, was expressed in INS-1 cells under the control of a doxycycline- inducible promoter by stable transfection. Two clones were found to exhibit dose-dependent, doxycycline-inducible expression of STAT5Δ749 and suppression of hGH-stimulated transcriptional activation of a STAT5-regulated PRL receptor (PRLR) promoter-reporter construct. Furthermore, induction of STAT5Δ749 expression completely inhibited hGH-induced DNA synthesis. Analysis of endogenous gene expression revealed a doxycycline-dependent inhibition of hGH-stimulated PRLR and cyclin D2 mRNA levels. Our results suggest that GH/PRL-induced β-cell proliferation is dependent on the Janus Kinase2 (JAK2)/STAT5 signaling pathway but not the MAPK, PI3K, and PKC signaling pathways. Furthermore, the cell cycle regulator cyclin D2 may be a crucial target gene for STAT5 in this process.