Use of the immunosuppressants calcineurin inhibitors (CNIs) are limited by irreversible kidney damage, hallmarked by renal fibrosis. CNIs directly damage many renal cell types. Given the diverse renal cell populations, additional targeted cell types and signaling mechanisms warrant further investigation. We hypothesize that fibroblasts contribute to CNI-induced renal fibrosis and propagate profibrotic effects via the TGFβ/Smad signaling axis. To test this, kidney-damage resistant mice (C57BL/6) received tacrolimus (10mg/kg) or vehicle for 21 days. Renal damage markers and signaling mediators were assessed. To investigate their role in renal damage, mouse renal fibroblasts were exposed to tacrolimus (1nM) or vehicle for 24 hrs. Morphological and functional changes in addition to downstream signaling events were assessed. Tacrolimus-treated kidneys displayed evidence of renal fibrosis. Moreover, α-SMA expression was significantly increased, suggesting the presence of fibroblast activation. TGFβ receptor activation and downstream Smad2/3 signaling was also upregulated. Consistent with in vivo findings, tacrolimus-treated renal fibroblasts displayed a phenotypic switch known as fibroblast-to-myofibroblast transition (FMT), as α-SMA, actin stress fibers, cell motility and Col IV expression were significantly increased. These findings were accompanied by concomitant induction of TGFβ signaling. Pharmacological inhibition of Smad2/3 activation attenuated tacrolimus-induced phenotypic changes. These findings suggest that 1) tacrolimus inhibits calcineurin (Cn) activity/NFAT activation while inducing TGFβ ligand secretion and receptor activation in the renal fibroblast, 2) aberrant TGFβ receptor activation stimulates Smad-mediated production of myofibroblast markers, promoting FMT and 3) FMT contributes to extracellular matrix (ECM) expansion in tacrolimus-induced renal fibrosis. These results incorporate renal fibroblasts to the growing list of CNI-targeted cell types and establish renal FMT as a process mediated via a TGFβ-dependent mechanism.