The present study investigated the effect of dietary Zn deficiency during sexual maturation on sperm integrity and testis phospholipid fatty acid composition. Male weanling Sprague–Dawley rats were randomised into four dietary groups for 3 weeks: Zn control (ZC; 30 mg Zn/kg); Zn marginally deficient (ZMD; 9 mg Zn/kg); Zn deficient (ZD; < 1 mg Zn/kg); pair fed (PF; 30 mg Zn/kg) to the ZD group. Morphology of cauda epididymal sperm and lipid profiles of testis phospholipids were analysed. The rats fed the ZD diet had a lower testis weight (P < 0·02). Seminal vesicles and prostate weight were also lower in the ZD and PF groups. Rats fed the ZD diet, but not the ZMD diet, had 34–35 % more abnormal spermatozoa and 24 % shorter sperm tail length than the ZC and PF rats (P < 0·001). Testis cholesterol concentration was higher in the ZD rats compared with the ZC and PF rats (P < 0·04). Testes were highly enriched with n-6 fatty acids by showing n-6 : n-3 fatty acid ratios of 27:1 in phosphatidylcholine (PC) and 23:1 in phosphatidylethanolamine (PE). The dominant fatty acid in testes was docosapentaenoic acid (22 : 5n-6), comprising 15 and 24 % of PC and PE, respectively. This fatty acid was significantly lower in the ZD rats, whereas 18 : 2n-6 was higher compared with the rats in the other diet groups. These results demonstrate that severe Zn deficiency adversely affects sperm integrity and modulates testis fatty acid composition by interrupting essential fatty acid metabolism. This suggests that Zn deficiency-associated abnormal testicular function is perhaps preceded by altered membrane fatty acid composition, especially of a major fatty acid, 22 : 5n-6.