Apolipoproteins A1 (ApoA1), an important protein in initiating sperm capacitation, has been found in the oviduct liquid, whilst whether it expresses in human spermatozoa has not yet been defined. ApoA1 expression was detected by reverse transcription–polymerase chain reaction, Western blot and immunofluorescence in sperm cells. Sperms were incubated with different concentration of ApoA1 antibody to observe the changes of sperm motility and apoptosis including annexin V binding to the cell surface, mitochondrial membrane potential and ultrastructural changes. In addition, cholesterol levels of human spermatozoa and fertilization in vitro were completed to explore the role of ApoA1 in fertilization process. We identify ApoA1 mRNA is presented in spermatozoa, and the ApoA1 protein, the molecular weights of 31 kD, is located at the postacrosomal region and neck of human spermatozoa. ApoA1 antibody affects cholesterol efflux of human spermatozoa and inhibits fertilization in vitro. Moreover, ApoA1 antibody decreases sperm motility and increases sperm apoptosis. This work shows ApoA1 plays an important role in maintaining cholesterol stability and sperm motility, survival rate and fertilization process. Furthermore, ApoA1 implications in the clinical work and other reproductive areas are needed to be studied.