Dietary vitamin E supplementation is beneficial to semen quality in different sheep and goat breeds. The aim of this research was to further investigate the effect of vitamin E in sheep on spermatogenesis and its regulatory mechanisms using RNA-seq. Thirty male Hu lambs were randomly divided into three groups. The animals received 0, 200 or 2000 IU/day vitamin E dietary supplementation for 105 days, and its effects were subsequently evaluated. The results indicate vitamin E supplementation increased the number of germ cells in the testes and epididymides. The positive effects were reduced, however, in animals that received 2000 IU/d vitamin E. Using the RNA-seq procedure, there was detection of a number of differentially expressed genes such as NDRG1, FSCN3 and CYP26B1 with these genes being mainly related to the regulation of spermatogenesis. Supplementation with 2000 IU/d vitamin E supplementation resulted in a lesser abundance of skeleton-related transcripts such as TUBB, VIM and different subtypes of collagen, and there was also an effect on the ECM-receptor interaction pathway. These changes appear to be responsible for the lesser beneficial effect of the greater vitamin E concentrations. The results provide a novel insight into the regulation of spermatogenesis by vitamin E at the molecular level, however, for a precise understanding of functions of the affected genes there needs to be further study.