The septin gene belongs to a highly conserved family of polymerizing GTP-binding cytoskeletal proteins. SEPTs perform cytoskeletal remodeling, cell polarity, membrane compartmentalization, mitosis, and vesicle trafficking by interacting with various cytoskeletons (e.g., tubulins, actins, and myosins). Septins have been implicated in the pathology of numerous diseases, including Alzheimer’s disease, hereditary neuralgic amyotrophy, leukemia, ovarian tumors, breast cancer, and male infertility. In mammalian spermatogenesis, the SEPT1/4/6/7 complex compartmentalizes between the mid-piece and the principal piece regions of the sperm tail, and this complex is important for maintaining a proper mitochondrial architecture of the spermatozoa mid-piece. Our previous studies have indicated that SEPT12 is expressed in post-meiotic male germ cells. The generated Septin12^{+/+/ +/-} chimeras with a Septin12 mutant allele deleted in the exons encoding the N-terminal GTP-binding domain were infertile. Spermatozoa from these chimeric mice showed abnormal sperm morphology (round head, acrosomal defects, and a bent tail), immotile sperm, maturation arrest, increased apoptosis, and a decreased sperm count. However, the mechanisms of reduced SEPT12 influences spermiogenesis are still unknown. We show how SEPT12 interacts and co-localizes with α- and β-tubulins, major cytoskeletons participating in manchette formation during mouse spermiogenesis. In Septin12 ^{+/+/ +/-} chimeric mice, manchette organization and tail development were disrupted. In cells, the knockdown of the SEPTIN12 transcript disrupts α- and β-tubulin organization. We discovered that the SEPTIN12-microtubulin complex is critical for manchette and tail formation during spermiogenesis.