Mammalian hibernation is a state of dormancy that is used by some animals to survive through the unfavorable conditions of winter, and is characterized by coordinated suppression of basal metabolism that is supported by global inhibition of energy/ATP-consuming processes. In this study, we examine the regulation of the anti-proliferatory TGF-β/Smad transcription factor signaling pathway in the liver tissue of the hibernating 13-lined ground squirrel Ictidomys tridecemlineatus. The TGF-β/Smad signaling pathway is known to mediate cell cycle arrest through induction of cell cycle dependent kinase inhibitors, and more recently, has been shown to regulate a wide range of cellular processes via its control of microRNA biosynthesis. We show that phosphorylation levels of the Smad3 protein at its activation residue is increased by ~1.5-fold during torpor, and this is associated with an increase in nuclear localization and DNA binding activity of Smad3. Expression levels of several TGF-β induced microRNAs previously described in human cells were also activated in ground squirrel during torpor. Among these were miR-21, miR-23a, and miR-107, which contain either the conserved R-SBE or R-SBE related motif found in microRNAs that are post-transcriptionally processed by Smad proteins. We show that levels of miR-21 were highly elevated at multiple stages of torpor, and predicted gene targets of miR-21 were enriched to multiple pro-growth cellular processes. Overall, we provide evidence that show the Smad3 transcription factor is activated in ground squirrels during torpor, and suggest a role for this signaling pathway in mediating anti-proliferatory signals via its transcriptional control of cell cycle inhibitors and downstream microRNAs.