The aim of the study was to evaluate the seasonality of andrological characteristics and hormonal profile of captive maned wolves (Chrysocyon brachyurus, Illiger 1811). Three adult males were evaluated from the Companhia Brasileira de Metalurgia e Mineração Scientific Breeding Center in Araxá, MG, Brazil, over 13 months. Semen was collected 2–3 times weekly and analysed. Scrotal circumference, biometrics and testicular volume were measured. Stool samples were collected 2–3 times weekly to analyse corticosteroid and testosterone metabolite concentrations. A success rate of 100% was achieved in the collection attempts during the breeding season (BS) and 77.8% during the non-breeding season (NBS). The interval to achieve penile erection was 1–5 min in the BS and 6–10 in the NBS (p < 0.001). Of the ejaculates collected, 80.0% contained sperm during BS, while 28.6% did during the NBS. The ejaculate had only one fraction, was odourless, predominantly translucent (72.4%), with a watery appearance, pH 6.7 and osmolarity of 352.8 mOsmol. Seasonal influences were seen in ejaculate volume (1.3 ml vs 0.4 ml), number of spermatozoa per ejaculate (73.9 × 106 vs 6.1 × 106) and percentage of live sperm (82.0% vs 66.1%) between the BS and NBS (p < 0.05), respectively. A high percentage of major sperm defects were observed in both seasons (50.1% in BS; 65.7% in NBS). Testicular volume was larger (p < 0.05; right testicles 13.1 cm3 in BS vs 4.0 cm3 in NBS, while left testicles 12.9 cm3 in BS vs 5.3 cm3 in NBS) and testicular consistency increased in the BS. No difference was seen in the basal faecal metabolite concentrations of testosterone; however, the corticosteroid concentrations were higher in the BS. Based on these results, it is possible to conclude that the collection of semen is feasible in captive maned wolves without compromising libido, seminal characteristics and reproductive behaviour and that sperm production is influenced by seasonality; however, it appears that there is no seasonal influence on basal testosterone concentrations.