Unlike humans and other mammals, zebrafish demonstrate a remarkable capacity to regenerate their injured hearts throughout life. Mitochondrial fatty acid β-oxidation (FAO) contributes to major energy demands of the adult hearts under physiological conditions; however, its functions in regulating cardiac regeneration and the underlying mechanisms are not completely understood. Different strategies targeting FAO have yield mixed outcomes. Here, we demonstrated that pharmacological inhibition of mitochondrial FAO with mildronate (MD) caused lipid accumulation in zebrafish larvae and suppressed ventricle regeneration. MD treatment impeded cardiogenic factor reactivation and cardiomyocyte (CM) proliferation, and impaired ventricle regeneration could be rescued by exogenous l-carnitine supplementation. Moreover, compared with the ablated hearts of wild-type fish, ventricle regeneration, cardiogenic factor reactivation and CM proliferation were significantly blocked in the ablated hearts of carnitine palmitoyltransferase-1b (cpt1b) knockout zebrafish. Further experiments suggested that NF-κB signaling and increased inflammation may be involved in the impediment of ventricle regeneration caused by systemic mitochondrial FAO inhibition. Overall, our study demonstrates the essential roles of mitochondrial FAO in zebrafish ventricle regeneration and reaffirms the sophisticated and multifaceted roles of FAO in heart regeneration with regard to different injury models and means of FAO inhibition.