In our work, we studied isolated rat cardiac myocyte autofluorescence emitted in response to excitation by visible (blue-violet) light. The distribution of the autofluorescence signal showed similar localization patterns with mitochondrial marker Mitotracker orange in the same cells but did not match the distribution of membrane marker Merocyanine 540. Autofluorescence excitation-emission spectra of both cardiomyocytes and isolated mitochondria showed a peak near 440 nm (excitation) and 520 nm (emission), suggesting their common origin. We were able to qualitatively separate self-fluorescing subcellular structures with different emission spectra using linear unmixing algorithms on image and volumetric λ stacks. We tested the hypothesis that autofluorescence can be modulated using different modulators of metabolism and respiration. Autofluorescence was increased by medium-chain fatty-acid octanoate but remained unchanged after the addition of lactate and pyruvate. Cyanide reduced the autofluorescence, while 2,4-dinitrophenol provoked its increase. These results indicate that changes in the levels of autofluorescence are not likely to arise from modification of the cytosolic redox state but rather result from actors of the mitochondrial redox state. We propose to further explore the spectroscopic properties of naturally occurring autofluorescence with the aim of their future use as a valuable tool for studying the metabolism and mitochondrial dysfunction of living cardiac cells.