Delta-6 desaturase-null mice ((-/-)) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The (-/-) males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type ((+/+)) and (-/-) males at weaning until 16 weeks of age (n = 3-5). A breeding success rate of DHA-supplemented (-/-) was comparable to (+/+). DHA-fed (-/-) showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed (-/-), but DPAn6 remained depleted. In AA-fed (-/-), AA was restored at the (+/+) level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of (+/+) and DHA-fed (-/-), whereas it diminished in (-/-) and AA-fed (-/-), suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.