During maturation, the surface of mammalian spermatozoa undergoes dramatic changes leading to the acquisition of properties vital for survival and performance in the female reproductive tract. A prominent change is the addition to the sperm surface of an atypical β-defensin polypeptide. In primates, the β-defensin DEFB126 becomes adsorbed to the entire sperm surface as spermatozoa move through the epididymal duct. DEFB126 has a conserved β-defensin core and a unique long glycosylated peptide tail. The carbohydrates of this domain contribute substantially to the sperm glycocalyx. DEFB126 is critical for efficient transport of sperm in the female reproductive tract, preventing their recognition by the female immune system, and might facilitate the delivery of capacitated sperm to the site of fertilization. A newly discovered dinucleotide deletion in the human DEFB126 gene is unusually common in diverse populations and results in a null allele. Predictably, men who are homozygous for the deletion produce sperm with an altered glycocalyx and impaired function, and have reduced fertility. Insights into the biology of DEFB126 are contributing to a better understanding of reproductive fitness in humans, as well as the development of diagnostics and therapeutics for male infertility.