Severe heat exposure causes mitochondrial fragmentation and dysfunction, which contribute to the pathogenesis of heat-related illness. l-Citrulline is a naturally occurring amino acid and has been suggested to influence heat shock responses. This study aimed to test whether l-citrulline supplementation would preserve mitochondrial integrity and attenuate heat-induced skeletal muscle injury and elucidate the underlying mechanisms. At 37°C, l-citrulline (2 mM) increased mitochondrial elongation in mouse C2C12 myoblasts, a process associated with a reduction in mitochondrial fission protein Drp1 levels. Mechanistic studies revealed that l-citrulline increased cellular nitric oxide (NO) levels, but not S-nitrosylation of Drp1. l-Citrulline caused a decrease in phosphorylation of Drp1 at Ser 616 and an increase in phosphorylation of Drp1 at Ser 637, which resulted in a reduced mitochondrial localisation of Drp1. L-NAME, a non-selective NO synthase inhibitor, abolished the increase in l-citrulline-induced NO levels and inhibited Drp1 phosphorylation changes and mitochondrial elongation, which indicates the involvement of a NO-dependent pathway. Under 43°C heat stress conditions, l-citrulline prevented translocation of Drp1 to mitochondria, mitochondrial fragmentation and decreased membrane potential. Finally, l-citrulline pretreatment inhibited heat-induced reactive oxygen species overproduction, caspase 3/7 activation, apoptotic cell death and improved cell viability. NO inhibitor l-NAME abolished all the above protective effects of l-citrulline under heat stress. Our results suggest that l-citrulline prevents heat-induced mitochondrial dysfunction and cell injury through NO-mediated Drp1 inhibition in C2C12 myoblasts. l-Citrulline may be an effective treatment for heat-related illnesses and other mitochondrial diseases.