Reducing temperature below the optimum of most vertebrate hearts impairs contractility and reduces organ function. However, a number of fish species, including the rainbow trout, can seasonally acclimate to low temperature. Such ability requires modification of physiological systems to compensate for the thermodynamic effects of temperature on biological processes. The current study tested the hypothesis that rainbow trout compensate for the direct effect of cold temperature by increasing cardiac contractility during cold acclimation. We examined cardiac contractility, following thermal acclimation (4 °C, 11 °C and 17 °C), by measuring the Ca2+ sensitivity of force generation by chemically skinned cardiac trabeculae as well as ventricular pressure generation using a modified Langendorff preparation. We demonstrate, for the first time, that the Ca2+ sensitivity of force generation was significantly higher in cardiac trabeculae from 4 °C-acclimated trout compared to those acclimated to 11 °C or 17 °C and that this functional change occurred in parallel with a decrease in the level of cardiac troponin T phosphorylation. In addition, we show that the magnitude and rate of ventricular pressure generation was greater in hearts from trout acclimated to 4 °C compared to those from animals acclimated to 11 or 17 °C. Taken together these results suggest that enhanced myofilament function, caused by modification of existing contractile proteins, is at least partially responsible for the observed increase in pressure generation after acclimation to 4 °C. In addition, by examining the phenotypic plasticity of a comparative model we have identified a strategy, used in vivo, by which the force generating capacity of cardiac muscle can be increased.