Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.