For human spermatozoa to successfully fertilize the oocyte, they need to undergo a timely acrosome reaction (AR). Factors which disturb the AR may lead to fertilization failure. The objective of this study was to investigate the effects of two cytokines namely tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) on the spontaneous, calcium ionophore-induced and progesterone-induced human sperm AR. Twenty-two normal semen samples were treated with increasing concentrations of TNF-α and IL-6 after spermatozoa were isolated by a double wash swim-up method. The AR was induced by calcium ionophore A23187 and progesterone. The AR was determined by using fluorescein isothiacyanate Pisum sativum agglutinin and observed under fluorescence microscope. Both TNF-α and IL-6 could decrease the spontaneous, ionophore and progesterone-induced AR (p < 0.05) in a dose-dependent manner. TNF-α showed a more potent inhibiting effect than IL-6 by inhibiting the AR at lower concentrations. This study has demonstrated that TNF-α and IL-6 play a role in inhibiting both the non-physiological as well as physiologically elicited AR by calcium ionophore and progesterone respectively.