Cold storage of the cauda epididymis, a male reproductive organ, is an efficient means of transporting genetically modified mice, an alternative to live animal shipment. The fertility of cold-stored sperm decreases in a time-dependent manner. However, the cause of the reduction in the fertility of sperm after cold storage remains unclear. It is known that cholesterol efflux from the sperm membrane is a trigger of capacitation. The dysfunction of cholesterol efflux due to changes in the membrane state in low temperatures may explain the reduced fertility. In this study, we examined the fertility (fertilization rate, acrosome reaction, and motility) and the amount of cholesterol and membrane fluidity in cold-stored sperm after treatment with two cholesterol acceptors, bovine serum albumin (BSA), and methyl-beta-cyclodextrin (MBCD). MBCD-treated sperm exhibited the highest rate of fertilization. Two-cell embryos derived from in vitro fertilization using 0.75-mM-MBCD-treated sperm after cold storage for 72 h developed to offspring. MBCD also displayed greater ability to remove cholesterol from the sperm membrane than BSA. The percentage of viable sperm with membrane destabilization was increased by MBCD in cold-stored sperm. The acrosome reaction strongly occurred in MBCD-treated sperm. In motility analysis, MBCD improved the lateral amplitude of head movement and beat frequency. These results suggest that MBCD-induced cholesterol efflux enhances membrane fluidity and promotes acrosome reaction and hyperactivation, resulting in improved fertility of cold-stored sperm.