BACKGROUND: Cytokines and hormones, including insulin, are known to modulate the hypothalamic-pituitary-testes axis and steroidogenesis, both centrally and peripherally. In the context of chronic inflammation and hyperinsulinaemia mediating male hypogonadism associated with obesity, metabolic syndrome and type 2 diabetes mellitus, these mechanisms are poorly understood and the impact of cytokines and insulin on Leydig cell steroidogenesis has not been fully elicited. This study aimed to further investigate the in vitro impact of TNFα, IL1ß, IL6, IL8 and insulin on Leydig cell function and steroidogenesis. METHODS: hCG-stimulated TM3 Leydig cells were exposed to various concentrations of TNFα, IL1ß, IL6, IL8 (100 ng/ml, 10 ng/ml, 1 ng/ml and 0.1 ng/ml) and insulin (10 ng/ml, 1 ng/ml, 0.1 ng/ml and 0.01 ng/ml) in optimal cell culture conditions over 48 h. Cell viability (XTT) and testosterone and progesterone concentrations (ELISA) were assessed using standardised laboratory techniques. RESULTS: TNFα significantly decreased cell viability and progesterone and testosterone concentrations in a dose-dependent relationship. IL1ß and IL6 had a subtle but significant negative effect on cell viability and testosterone concentrations, with a marked significant decrease in progesterone concentration at all concentrations investigated. IL8 showed an increase in cell viability, with no significant effect on testosterone concentrations alongside a significant decrease in progesterone concentrations. Insulin significantly increased cell viability and testosterone concentrations in a dose dependent relationship, but interestingly significantly decreased progesterone concentrations. CONCLUSIONS: The inflammatory cytokines TNFα, IL1β and IL6 cause a dose dependent decline in steroidogenesis in TM3 Leydig cells. These results suggest that chronic inflammation may downregulate steroidogenesis in males via direct modulation of Leydig cell function. However, IL8 may stimulate TM3 Leydig cell growth. Insulin is associated with a dose-dependent increase in testosterone synthesis, with a significant decline in progesterone synthesis. With the phenomenon of insulin resistance, the literature is unclear on the potential role of hyperinsulinaemia in steroidogenesis. Further studies are warranted in order to fully elicit the molecular mechanisms and interactions of these molecules on male steroidogenesis.