Many small mammals turn to hibernation to survive the winter, cycling through bouts of prolonged torpor where metabolic rate and body temperature fall to low levels. Remarkably, hypertrophy is promoted in cardiac muscle to support the stronger contractions needed in the cold. We proposed that altered expression of mRNA/protein levels of myocyte enhancer factor-2 (MEF2A, MEF2C) transcription factors and downstream targets (e.g., desmin, glucose transporter 4, and myomesin 1) would aid cardiac muscle of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) in meeting challenges associated with hibernation. Gene and protein responses were compared over six conditions: control (euthermic animals in a 5 °C cold room), entrance into torpor, short and long torpors, arousal and interbout. Mef2a relative transcript levels were significantly elevated from controls contributing to increases in MEF2A protein levels throughout the torpor–arousal bout. In addition, levels of phosphorylated, activated MEF2A (Thr312) correlated with increases in MEF2A–DNA binding. MEF2C transcript/protein levels were significantly elevated over controls at selected sampling points whereas phosphorylated/activated MEF2C (Ser387) levels rose during torpor and DNA binding was most prominent during entrance into torpor. Some gene targets of MEF2 action were also upregulated. Desmin transcript levels remained constant whereas enhanced protein expression occurred during entrance into torpor. Glut4 transcript levels were enhanced in arousal and protein expression was elevated over all five sampling points during torpor/arousal. Myomesin 1 transcript levels increased between early torpor and early arousal and protein levels increased during entrance and deep torpor. These data provide insights into the changes in gene/protein in expression that help to prepare cardiac muscle for hibernation.