To examine the roles of mitochondrial calcium Ca ([Ca]) and cytosolic Ca ([Ca]) in the regulation of hepatic mitochondrial fat oxidation, we studied a liver-specific mitochondrial calcium uniporter knockout (MCU KO) mouse model with reduced [Ca] and increased [Ca] content. Despite decreased [Ca], deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux, α-ketoglutarate dehydrogenase flux, and succinate dehydrogenase flux in vivo. Rates of [C]palmitate oxidation and intrahepatic lipolysis were increased in MCU KO liver slices, which led to decreased hepatic triacylglycerol content. These effects were recapitulated with activation of CAMKII and abrogated with CAMKII knockdown, demonstrating that [Ca] activation of CAMKII may be the primary mechanism by which MCU deletion promotes increased hepatic mitochondrial oxidation. Together, these data demonstrate that hepatic mitochondrial oxidation can be dissociated from [Ca] and reveal a key role for [Ca] in the regulation of hepatic fat mitochondrial oxidation, intrahepatic lipolysis, gluconeogenesis, and lipid accumulation.