Mitochondrial fatty acid oxidation (FAO) has been proposed to be a major bioenergetic pathway in prostate cancer. However, this concept fails to consider FAO relative to other mitochondrial substrates. Here, we found extracellular long-chain fatty acids (LCFAs), including palmitate, stearate, oleate, linoleate, linolenate, are minor sources of carbon entering the TCA cycle compared to glucose and glutamine in prostate cancer cells, despite being assimilated in the mitochondria as acyl-carnitines. In contrast, cardiolipins were a prominent LCFAs sink, with some species achieving greater than 50% 13C-labelling within 6 hours, suggesting high cardiolipin turnover using extracellular LCFAs. Knockdown of CPT1a, the rate-limiting enzyme of LCFA entry into mitochondria, reduced the incorporation of extracellular linoleate into cardiolipins. These results demonstrate that FAO is not a major input for the TCA cycle and provide evidence for an underappreciated role for CPT1a in regulating LCFAs entry into mitochondria for cardiolipin remodelling.