Updated project metadata. Globozoospermia is a severe teratozoospermia characterized by round-headed spermatozoa without acrosomes, resulting in male infertility. The clinical treatment of globozoospermia involves primarily intracytoplasmic sperm injection because of the low fertilization rate. The deletion of Dpy19l2 is known to be the major cause of globozoospermia. The defects in Dpy19l2-deficient human sperm at the protein level were characterized by performing a isotope-based quantitative proteomic analysis between Dpy19l2-deficient globozoospermic spermatozoa and normal controls. A total of 2,549 proteins were identified, including 495 differentially expressed proteins (fold-change >2), of which 322 were upregulated and 123 were downregulated. The levels of several important proteins, including SPACA 1, IZUMO1, ZPBP1, and PLCZ1, were decreased in globozoospermic sperm. Bioinformatics analysis indicated the Dpy19l2-deficient sperm presented molecular defects in acrosome, chromatin, sperm-egg interaction, and fertilization. This study may provide insights into the mechanism of globozoospermia and help develop better treatments to increase the success rate of fertilization.