Hibernation is an adaptive strategy used by various mammals to survive the winter under situations of low ambient temperatures and limited or no food availability. The heart of hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) has the remarkable ability to descend to low, near 0°C temperatures without falling into cardiac arrest. We hypothesized that the transcription factors GATA4 and Nkx2-5 may play a role in cardioprotection by facilitating the expression of key downstream targets such as troponin I, troponin C, and ANP (atrial natriuretic peptide). This study measured relative changes in transcript levels, protein levels, protein post-translational modifications, and transcription factor binding over six stages: euthermic control (EC), entrance into torpor (EN), early torpor (ET), late torpor (LT), early arousal (EA), and interbout arousal (IA). We found differential regulation of GATA4 whereby transcript/protein expression, post-translational modification (phosphorylation of serine 261), and DNA binding were enhanced during the transitory phases (entrance and arousal) of hibernation. Activation of GATA4 was paired with increases in cardiac troponin I, troponin C and ANP protein levels during entrance, while increases in p-GATA4 DNA binding during early arousal was paired with decreases in troponin I and no changes in troponin C and ANP protein levels. Unlike its binding partner, the relative mRNA/protein expression and DNA binding of Nkx2-5 did not change during hibernation. This suggests that either Nkx2-5 does not play a substantial role or other regulatory mechanisms not presently studied (e.g. posttranslational modifications) are important during hibernation. The data suggest a significant role for GATA4-mediated gene transcription in the differential regulation of genes which aid cardiac-specific challenges associated with torpor-arousal.