Osteoclasts specialize in bone resorption and are critical for bone remodeling. Previous studies have shown that osteoclasts possess abundant mitochondria and derive most energy through oxidative phosphorylation (OXPHOS). However, the energy substrates fueling OXPHOS in osteoclasts remain to be fully defined. Here, we showed that osteoclast differentiation was coupled with increased oxidation of glucose, glutamine, and oleate. Transcriptomic analyses with RNA sequencing revealed marked upregulation of genes participating in OXPHOS and mitochondrial fatty acid oxidation, during osteoclast differentiation. Increased mitochondrial oxidation of long-chain fatty acids was required for osteoclast differentiation in vitro. However, blocking fatty acid oxidation in vivo, by deletion of carnitine palmitoyltransferase 1a (Cpt1a) in osteoclast progenitors, impaired osteoclast formation only in the female mice. The Cpt1a-deficient females were further protected from osteoclast activation by a high-fat diet. The males, on the contrary, exhibited normal bone resorption despite Cpt1a deletion, regardless of the dietary fat content. Moreover, concurrent deletion of mitochondrial pyruvate carrier 1 and Cpt1a, blocking mitochondrial oxidation of both glucose and fatty acids in the osteoclast lineage, failed to impede bone resorption in the males. The study therefore uncovers a female-specific dependence on mitochondrial oxidation of fatty acids and glucose in osteoclasts in vivo.