Septins are polymerizing GTP binding proteins required for cortical organization during cytokinesis and other cellular processes. A mammalian septin gene Sept4 is expressed mainly in postmitotic neural cells and postmeiotic male germ cells. In mouse and human spermatozoa, SEPT4 and other septins are found in the annulus, a cortical ring which separates the middle and principal pieces. Sept4-/- male mice are sterile due to defective morphology and motility of the sperm flagellum. In Sept4 null spermatozoa, the annulus is replaced by a fragile segment lacking cortical material, beneath which kinesin-mediated intraflagellar transport stalls. The sterility is rescued by injection of sperm into oocytes, demonstrating that each Sept4 null spermatozoon carries an intact haploid genome. The annulus/septin ring is also disorganized in spermatozoa from a subset of human patients with asthenospermia syndrome. Thus, cortical organization based on circular assembly of the septin cytoskeleton is essential for the structural and mechanical integrity of mammalian spermatozoa.