Mammary pathogenic Escherichia coli (MPEC) causes mastitis, which results in substantial economic losses to the dairy industry. A high percentage of Escherichia coli isolated from cows with clinical mastitis harbor adhesin genes, such as fimH. However, it is unclear whether these adhesins are important in the adhesion of MPEC to bovine mammary epithelial cells (BMECs). Therefore, we investigated the effect of adhesins (EcpD, FdeC, and FimH) in MPEC on adherence to the bovine mammary epithelium using cultured BMECs. For this purpose, we used wild-type MPEC as well as single- and double-mutants of fimH, ecpD, and fdeC, and performed adhesion assays with BMECs. First, BMECs were cultured in the presence of lactogenic hormones to induce milk component production and tight junction formation. The bacterial count of the wild-type strain that adhered to the BMECs increased in a dose-dependent manner. In deletion mutant strains, the ΔfimH strain showed lower adhesion (P < 0.05), whereas the adhesion ratio of the ΔecpD and ΔfdeC strains was not statistically different compared with that of the wild-type strain (P > 0.05). Additionally, the fimH/fdeC double-deletion mutants showed the lowest adhesion to BMECs. In conclusion, FimH is crucial in the adhesion of MPEC to BMECs. Overall, our work identifies FimH or FimH/FdeC as interesting targets for future drugs or vaccines to improve the treatment, prevention or chronicity of mastitis induced by MPEC.