Dietary carotenoids have a high antioxidant capacity, so it has been hypothesised that carotenoid supplimentation will improve sperm production and quality by protecting sperm from oxidative damage. The effects of carotenoids on sperm have only been assessed in three vertebrate species, and evidence for improved sperm concentration and motility remains equivocal. One reason for this might be that in most studies there has not been an assessment of the effects of single carotenoid compounds over a range of doses. Applied research focused on developing ways to improve sperm quality could benefit the captive breeding and conservation of threatened species. The aim of the present study was to assess a dose-dependent effect of beta-carotene supplementation on sperm concentration and motility in the endangered booroolong frog (Litoria booroolongensis). Individuals were supplemented with one of four beta-carotene doses (0, 0.1, 1 and 10 mg/g) from hatching until sexual maturity (53 weeks). Sperm concentration was determined prior to activation, and percent sperm motility and sperm velocity were measured at 0, 3 and 6 h post-activation using computer-assisted sperm analysis. Unexpectedly, beta-carotene had no significant effect on sperm concentration, percent motility or velocity at any time point, providing no evidence for beneficial effects. Findings of the present study indicate there are likely to be species-specific differences in sperm production and motility that influence the risk of oxidative damage to sperm and dependence on dietary antioxidants to inhibit these detrimental effects.