Transglutaminase-1 (TGase-1) is a Ca(2+)-dependent enzyme capable of cross-linking a variety of proteins and promoting wound healing in the skin. In this study, we examined the role of TGase-1 in proliferation of renal proximal tubular cells (RPTC). TGase-1, but not TGase-2, -5, and -7, was expressed in RPTC. Treatment with monodansylcadarevine (MDC), a selective TGase inhibitor or down-regulation of TGase-1 with small interfering RNA (siRNA) decreased RPTC proliferation. Proliferation of RPTC was accompanied by activation of Akt and Stat-3 (signal transducer and activator of transcription-3). Treatment with MDC or TGase-1 siRNA decreased Stat-3 but not Akt phosphorylation. Further studies showed that the Janus-activated kinase 2 (JAK2) mediates phosphorylation of Stat-3, and knockdown of either JAK2 or Stat-3 by siRNA decreased RPTC proliferation. However, inhibition of TGase-1 decreased phosphorylation of Stat-3 but not JAK2. Overexpression of Stat-3, JAK2, and/or TGase-1 in RPTC revealed that JAK2 is indispensable for TGase-1 to induce Stat-3 phosphorylation and TGase-1 potentiates JAK2-induced Stat-3 phosphorylation. Consistent with these observations, we found that inhibition of TGase-1 and the JAK2-Stat-3 signaling pathway decreased the transcriptional activity of Stat-3 and expression of the Stat-3-targeted genes, cyclin D1 and cyclin E. Conversely, overexpresssion of TGase-1 enhanced the JAK2-dependent transcriptional activity of Stat-3. Finally, TGase-1 was found to interact with JAK2, and this interaction was inhibited by MDC. These results demonstrate that TGase-1 plays an important role in regulation of renal epithelial cell proliferation through the JAK2-Stat-3 signaling pathway.