Vitamin E was discovered for its implication in reproductive biology, and its transport in mammalian plasma and brain was shown to be governed by plasma phospholipid transfer protein (PLTP). We show that PLTP deficiency is associated with hypofertility of mouse males but not mouse females, and it accounts for a significant decrease in total number of pups produced over a 2-month breeding period of PLTP knocked out mice (-32%, P<0.03). PLTP is highly expressed in epididymis of mouse males, and alpha-tocopherol, the main vitamin E isomer in vivo, was significantly less abundant in cauda and caput epididymis of PLTP-deficient mice as compared with wild-type counterparts (caput: -26%, P<0.05; cauda: -21%, P<0.05). Mature spermatozoa from PLTP-deficient epididymis were shown to retain an abnormal alpha-tocopherol content. PLTP deficiency tended to reduce sperm motility as shown by a 24% reduction in spermatozoa with progressive motility (P<0.02), with no change in other sperm parameters as compared with wild-type males. Finally, in vitro fertilization rates of wild-type oocytes with spermatozoa from PLTP-deficient males were markedly reduced as compared with those measured with spermatozoa from wild-type males (-60%, P<0.05). It is concluded that PLTP is a new, key factor that determines sperm motility and male fertility.