Mycoplasma synoviae (MS) is an important pathogen that can cause respiratory diseases in poultry and birds, leading to serious economic losses in the world and impacting the development of the poultry industry. However, the pathogenic mechanisms of MS infection are still unclear, resulting in a lack of effective diagnosis and treatment strategies. This study aimed to uncover the infection effect caused by MS in chicken oviduct cells. MS-infected chicken oviduct cells with different infection times and doses were collected, and DAPI staining was performed to monitor the morphological changes of cell nuclei. Cell viability was detect by CCK-8 assay. The expression of marker genes of cell apoptosis was determined by RT-qPCR and Western blotting assay, respectively. The results showed that MS infection significantly inhibited the growth of chicken oviduct cells, caused the nuclear shrinkage and rupture, activated the expression of apoptosis marker genes Caspase 3 and Beclin-1, and increased the apoptosis rate. These results indicated that MS infection inhibits cell proliferation and promotes apoptosis in chicken oviduct cells, which provided a basis for further revealing the MS pathogenic mechanism and provided a foundation for the future development of anti-infection strategies.