Spermatozoa produce increasing levels of reactive oxygen species (ROS) during development. We examined the functional organisation of NOX‐2 subunits in developing spermatozoa and its involvement in ROS generation. We found NOX‐2 components associated predominantly with caveolin‐rich microdomains of sperm head and mid piece membranes. Capacitation in vitro enriched NOX‐2 components within the caveolin‐positive sperm membrane rafts. Progesterone, but not 17β‐oestradiol, brought about loss of caveolin and extensive reorganisations in NOX‐2 distribution on sperm head microdomains and accompanying activation in the levels of superoxide. The sensitivity of superoxide production by spermatozoa to methyl‐β‐cyclodextrin confirms that NOX‐2 function in spermatozoa is tightly dependent on raft organisation. We conclude that progesterone‐induced reorganisation of NOX‐2 components in sperm membrane rafts is critical for sperm functioning.