Sheep serum is commonly supplemented to capacitating media for ram spermatozoa in order to support in vitro fertilisation (IVF). Its exact role in capacitation is uncertain but it is thought to support cholesterol efflux owing to the presence of cholesterol acceptors such as albumin and the various subtypes of high density lipoproteins (HDLs). During cholesterol efflux, HDLs remove this sterol via a facilitated pathway that involves interaction with cholesterol transporters located in the plasma membrane, including ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B, type I (SR-BI). With this knowledge, the objectives of the current study were to i) determine if sheep serum or the specific cholesterol acceptor, HDLs, were able to elicit cholesterol efflux in ram spermatozoa and ii) whether the cholesterol transporters, ABCA1 and SR-B1 were functional during cholesterol efflux as assessed by the use of antagonists. Both sheep serum and HDLs were able to elicit cholesterol efflux alone by up to 40-50% (as measured with the BODIPY-cholesterol assay) and caused additional efflux when combined with 0.3% BSA. Surprisingly, the addition of HDLs to ram spermatozoa also induced hyperactivation in at least 19.2% (95% CI: 12.3%-25%) of the population, which was in contrast to the addition of BSA alone (2.9%; CI: 1.6%-3.9%). Following the inhibition of ABCA1 and SR-B1 by glibenclamide (both transporters) and valspodar (ABCA1 alone), sheep serum-mediated cholesterol efflux was reduced by 15% compared to when these inhibitors were absent. In contrast, only glibenclamide was able to reduce HDL-mediated cholesterol efflux. Together, these findings indicate that cholesterol efflux in ram spermatozoa may be regulated by a pathway involving the interaction between HDL subtypes and the cholesterol transporters, ABCA1 and SR-B1. Furthermore, the induction of hyperactivation under these conditions suggests a functional link with HDL-mediated cholesterol efflux.