Semen cryopreservation plays a critical role for a wide application of both AI and in vitro embryo production in buffalo. In this species, spermatozoa are more susceptible to hazards during freezing and thawing than cattle spermatozoa, thus resulting in lower fertilizing potential (Andrabi et al. 2008 Anim. Reprod. Sci. 104, 427–433). Carnitine is a quaternary ammonium compound with antioxidant capacities, able to reduce the availability of lipids for peroxidation by transporting fatty acids into the mitochondria for β-oxidation to generate adenosine triphosphate (ATP) energy (Tanphaichitr and Leelahagul 1993 Nutrition 9, 246–54). It is known that cryopreservation processes decreases the intracellular concentration of carnitine in spermatozoa (Reyes-Moreno et al. 2000 J. Androl. 21, 876–86). In cattle, supplementation of semen extender with carnitine improves sperm motility and DNA integrity (Bucak et al. 2010 Cryobiology 61, 248–53). The aim of this study was to evaluate whether supplementation of semen extender with carnitine would increase ATP content in buffalo sperm and affect post-thawing motility. Eight ejaculates from 4 bulls were used for the trial. Each ejaculate was split into 3 equal aliquots and diluted at 37°C with BioXcell extender containing 0 (control), 2.5, and 7.5 mm carnitine to a final concentration of 30 × 10⁶ spermatozoa/mL. After 4 h at 4°C, the straws were frozen in an automated system. At thawing, sperm motility was evaluated by phase contrast microscopy at 40× magnification (Gillan et al. 2008 Anim. Reprod. Sci. 103, 201–204). Adenosine triphosphate content was measured using a Colourimetric ATP Assay Kit (Biovision, Milpitas, CA, USA). Briefly, Percoll-separated spermatozoa were homogenised and then deproteinized using 10-kDa spin columns. Samples were incubated at RT for 30 min and absorbance was measured at 570 nm in a microplate reader. Differences in sperm motility and ATP content among groups were analysed by ANOVA. Both concentrations of carnitine increased post-thawing sperm motility compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6, respectively, with 0, 2.5, and 7.5 mm carnitine; P < 0.05). Interestingly, carnitine increased ATP content of buffalo frozen–thawed sperm in a dose-dependent manner (4.1 ± 0.1, 5.3 ± 0.1, and 8.2 ± 0.4 nm × 10⁸ sperm, respectively, with 0, 2.5, and 7.5 mm carnitine; P < 0.01). In conclusion, the enrichment of semen extender with carnitine improved post-thawing motility of buffalo sperm by boosting mitochondrial ATP production, hence providing energy for use by spermatozoa.