Through whole-exome sequencing of 800 patients with asthenozoospermia, we identified biallelic TEX44 variants in six individuals, all of whom exhibited defective mitochondrial sheath assembly and impaired sperm motility. Using Tex44 knockout mice, we demonstrated that TEX44 interacts with carnitine palmitoyltransferase 1B (CPT1B) to form a mitochondrial glue, anchoring adjacent mitochondria and facilitating the assembly of the sperm-specific mitochondrial sheath.