Abstract We aimed (I) to simulate an in vivo milieu, through establishing an in vitro paradigm to study sperm–oviductal interactions using different segments of oviduct, as well as different incubation media, and (II) to investigate spatial changes of oviductal gene expression. Two experiments were designed; one was to investigate the yield of oviduct aggregates from different oviduct segments; in the second experiment, we observed effects of different incubation media on sperm–oviductal binding. Oviduct cell pellets before (control) and after sperm binding were collected for RNA isolation and gene expression. Isthmus resulted in a higher aggregate yield and possessed the highest affinity towards spermatozoa. The different segments of oviduct showed clear changes in gene expression after sperm binding. TALP medium promoted formation of a higher number of oviduct aggregates towards spermatozoa. Different media resulted in profound alterations in isthmus gene expression. Collectively, isthmus segment in TALP media showed the highest binding affinity to spermatozoa. At the molecular level, our in vitro model was successful for simulation in vivo milieu. Thus, our findings could be used as a simple tool to gain more insights into the molecular regulation of sperm movement, selection and affinity for oviductal binding in buffaloes.