Mastitis remains one of the major diseases of dairy cattle, with important consequences on the economy of dairy farms, on animal welfare and on the use of antibiotics. Coliform bacteria, mainly E. coli, are responsible for most cases of severe mastitis of cattle worldwide. Because vaccination is a promising alternative to the use of antibiotics to treat mastitis, we intended to decipher the contribution of both cellular and humoral immunity in the response of cows to vaccination against E. coli mastitis. Cows were immunized with either the smooth strain E. coli P4, the rough strain E. coli J5 or recombinant OmpA. We assessed the opsonic activities in ex vivo phagocytosis assay after intramammary challenge with the homologous strain. The cell-dependent bactericidal activity of milk after challenge was not different in infected quarters from immunized versus control cows. Immune serum to J5 labeled at best a small subpopulation of live smooth bacteria, whereas the whole population of rough bacteria bound antibodies. Affinity-purified antibodies to OmpA labeled only a subpopulation of rough or smooth strains. We present evidence that the O-antigen shields outer membrane proteins. The pre-immune serum was able to opsonize all tested strains while the immune anti-J5 serum did not improve on this opsonic activity. These results show that immunization induced antibodies to antigens shared by most strains were ineffective at opsonizing smooth mastitis-associated E. coli strains. Our results support the role of naturally acquired antibodies as major opsonins, and question the usefulness of vaccine-induced antibodies for opsonophagocytosis.