Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. Vinca alkaloids such as vincristine (VCR), widely used in cancer treatment, are no exception, as their beneficial actions are often supplanted by resistance. In the present work we utilized HL-60 human leukemia cells and a VCR-resistant counterpart, HL-60/VCR, as a model to determine potential interplay between SL metabolism and mitochondrial bioenergetics supportive of multidrug resistance (MDR). Relative to wild-type cells, HL-60/VCR presented with global alterations in SL composition, typified by upregulated expression of sphingosine kinase (SPHK1), which catalyzes formation of sphingosine 1-phosphate (S1P), glucosylceramide synthase (GCS), which catalyzes formation of glucosylceramides (GC), and acid ceramidase, responsible for ceramide hydrolysis. In support of these changes, VCR resistance was also characterized by increases in S1P, several molecular species of ceramide and GC, and changes in sphingomyelin (SM) molecular species. With respect to mitochondria, despite increased basal respiration in HL-60/VCR cells, direct interrogation of the mitochondrial network revealed intrinsic respiratory complex insufficiency, largely localized to complex I (CI). Importantly, forced ceramide accumulation in wild-type cells phenocopied the respiratory insufficiency observed in HL-60/VCR, and co-targeting SL metabolism and CI induced synergistic cytotoxicity in HL-60/VCR cells, as well as in other MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.