Lactoferrin (Lf) may play a key role in the clearance of microorganisms from a host. To study in vitro the bactericidal mechanisms of Lf during nonlactating periods, we investigated whether the effects of Lf were influenced by bovine mammary gland secretory cells (MGSC) and fresh normal bovine serum (NBS) as a source of complement. Phagocytic killing tests demonstrated that a phagocytic mixture of unopsonized Staphylococcus aureus (S. aureus) and MGSC in the presence of Lf reduced bacterial growth, compared with that of unopsonized S. aureus and MGSC without Lf. The opsonization with Lf and fresh NBS together resulted in more than a 95% reduction in CFU. The activation of complement induced by Lf also resulted in increased deposition of C3 on S. aureus, and the phagocytic activity of MGSC was augmented by opsonization with Lf and fresh NBS. Inhibition of C3 deposition by Lf was not induced in the presence of Mg‐EGTA, but was induced by the addition of bovine Lf antiserum. These results strongly suggest that Lf induces the activation of complement in fresh NBS mainly through an alternative pathway. The results demonstrated a Lf‐dependent, antibody‐independent and complement‐mediated phagocytic killing of S. aureus, and implied that Lf was synergistically capable of activating both the alternative pathway of the bovine complement cascade and phagocytosis by phagocytes.