The Western diet can lead to alterations in cardiac function and increase cardiovascular risk, which can be reproduced in animal models by implementing a high-fat diet (HFD). However, the mechanism of these alterations is not fully understood and may be dependent on alterations in heart lipid composition. The aim of this study was to evaluate the effect of an HFD on the fatty acid (FA) composition of total lipids, as well as of various lipid fractions in the heart, and on heart function. C57BL/6 mice were fed an HFD or standard laboratory diet. The FA composition of chow, serum, heart and skeletal muscle tissues was measured by gas chromatography–mass spectrometry. Cardiac function was evaluated by ultrasonography. Our results showed an unexpected increase in polyunsaturated FAs (PUFAs) and a significant decrease in monounsaturated FAs (MUFAs) in the heart tissue of mice fed the HFD. For comparison, no such effects were observed in skeletal muscle or serum samples. Furthermore, we found that the largest increase in PUFAs was in the sphingolipid fraction, whereas the largest decrease in MUFAs was in the phospholipid and sphingomyelin fractions. The hearts of mice fed an HFD had an increased content of triacylglycerols. Moreover, the HFD treatment altered aortic flow pattern. We did not find significant changes in heart mass or oxidative stress markers between mice fed the HFD and standard diet. The above results suggest that alterations in FA composition in the heart may contribute to deterioration of heart function. A possible mechanism of this phenomenon is the alteration of sphingolipids and phospholipids in the fatty acid profile, which may change the physical properties of these lipids. Since phospho- and sphingolipids are the major components of cell membranes, alterations in their structures in heart cells can result in changes in cell membrane properties.