Thermal acclimation can alter cardiac function and morphology in a number of fish species, but little is known about the regulation of these changes. The purpose of the current study was to determine how cold acclimation affects zebrafish (Danio rerio) cardiac morphology, collagen composition, and connective tissue regulation. Heart volume, the thickness of the compact myocardium, collagen content, and collagen fiber composition were compared between control (27°C) and cold acclimated (20°C) zebrafish using serially sectioned hearts stained with picrosirius red. Collagen content and fiber composition of the pericardial membrane were also examined. Cold acclimation did not affect the volume of the contracted heart, however there was a significant decrease in the thickness of the compact myocardium. There was also a decrease in the collagen content of the compact myocardium and in amount of thick collagen fibers throughout the heart. Cold-acclimated zebrafish also increased expression of the gene transcript for matrix metalloproteinase 2, matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2, and collagen Type 1 α1. We propose that the reduction in the thickness of the compact myocardium as well as the change in collagen content may help to maintain the compliance of the ventricle as temperatures decrease. Together, these results clearly demonstrate that the zebrafish heart undergoes significant remodelling in response to cold acclimation.