The interactions between oviductal fluid (OF) proteins and spermatozoa play major roles in sperm selection, storage and capacitation before fertilization. However, only a few sperm-interacting proteins in the OF has been identified and very little is known about the regulation of sperm-oviduct interactions across the estrous cycle. Samples of bovine frozen-thawed sperm from three bulls were incubated with OF at pre-, post-ovulatory stages (Pre-/Post-ov) or luteal phase (LP) of the estrous cycle (7 mg/mL proteins, treated groups) or with a protein-free media (control). The proteomes of sperm cells were assessed by nanoLC-MS/MS and quantified by label-free methods. A total of 27 sperm-interacting proteins originating in the OF were identified. Among those, 14 were detected at all stages, eight at Post-ov and LP and five only at LP. The sperm-interacting proteins detected at all stages or at LP and Post-ov were on average more abundant at LP than at other stages (P < 0.05). At Pre-ov, OVGP1 was the most abundant sperm-interacting protein while at Post-ov, ACTB, HSP27, MYH9, MYH14 and OVGP1 were predominant. Different patterns of abundance of sperm-interacting proteins related to the stage were evidenced, which greatly differed from those previously reported in the bovine OF. In conclusion, this study highlights the important regulations of sperm-oviduct interactions across the estrous cycle and provides new protein candidates that may modulate sperm functions.