Abstract Heme is an essential cofactor and signaling molecule. All heme-dependent processes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins in virtually every subcellular compartment. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. To address this, we developed a pulse-chase assay in which, upon the initiation of heme synthesis, heme mobilization into the mitochondrial matrix, cytosol and nucleus is monitored using fluorescent heme sensors. Surprisingly, we found that heme trafficking to the nucleus occurs at a faster rate than to the matrix or cytosol. Further, we demonstrate that GTPases in control of mitochondrial fusion, Mgm1, and fission, Dnm1, are positive and negative regulators of mitochondrial-nuclear heme trafficking, respectively. We also find that heme controls mitochondrial network morphology. Altogether, our results indicate that mitochondrial dynamics and heme trafficking are integrally coupled.