During spermiogenesis, the manchette is an important structure for sperm head and tail formation. However, the mechanisms responsible for this process are poorly understood. In a previous study, we established a comparative proteome profile for mouse testis during the first wave of spermatogenesis, and provided lists of proteins of potential importance in the regulation of male fertility and infertility. Here we have selected Arl3, one of these interesting proteins, and investigated its expression and function during spermiogenesis. Western blotting was used to determine Arl3 expression levels in mice at different time points from birth to adulthood. The results show Arl3 was expressed from birth, and the expression level increased significantly from Week 4, when mouse spermiogenesis begins. Immunohistochemistry and indirect immunofluorescence were used to investigate the Arl3 expression during sperm development, and the intracellular localization of Arl3 in more detail. In elongating spermatids from steps 8 to 15, Arl3 was localized to the posterior section of the head, in a similar pattern to the manchette. The Arl3 signal was colocalized during spermiogenesis with α-tubulin, a marker for the manchette. To investigate the possible functional role of Arl3, mouse testes were injected with small interfering RNA (siRNA) against Arl3, or control siRNA. Western blotting showed a 60% reduction in the Arl3 expression after 72 h, and a significant increase in sperm abnormalities after 3 weeks compared with the negative control. In conclusion, we propose that Arl3 is a novel manchette-related protein with an important role in spermiogenesis.