Intrinsic factors such as proteins modulate the fertilising ability of male gametes. We compared detergent-extracted sperm protein composition of bulls with different fertility indexes in order to highlight putative fertility markers of sperm. Frozen semen from 23 Holstein bulls with documented fertility was used. According to their 'fertility solution' (SOL), as calculated by the Canadian dairy network, bulls were divided into four groups: high fertility (HF) (SOL>3.0; n=6), medium-HF (2.9>SOL>2.0; n=5), medium-low fertility (-2.8>SOL>-4.9; n=8) and low fertility (LF; SOL<-5.0; n=4), with a SOL=0 being the average. Triton X-100 protein extracts from ejaculated spermatozoa were subjected to two-dimensional difference gel electrophoresis, and polypeptide maps were quantitatively analysed by ImageMaster software. Nine protein spots showed significant differences between the HF and LF groups, and eight of these proteins were identified by liquid chromatography-tandem mass spectrometry. T-complex protein 1 subunits epsilon and (CCT5 and CCT8), two isoforms of epididymal sperm-binding protein E12 (ELSPBP1), proteasome subunit alpha type-6 and binder of sperm 1 (BSP1) were more expressed in the LF group than in the HF group. On the other hand, adenylate kinase isoenzyme 1 (AK1) and phosphatidylethanolamine-binding protein 1 (PEBP1) were more expressed in the HF group than in the LF group. The presence and expression level of ELSPBP1, BSP1, AK1 and PEBP1 were confirmed by western blot. A linear regression model established that CCT5 and AK1 explained 64% (P<0.001) of the fertility scores. The reported functions of these proteins are in agreement with a putative involvement in defective sperm physiology, where lower or higher levels can jeopardise sperm ability to reach and fertilise the oocyte.