It was previously reported that injection of anaflatoxin B1 (AnAFB1) conjugated to keyhole limpet hemocyanin (KLH), together with Freund's adjuvant, was effective in inducing in cows a long lasting titer of anti-aflatoxin B1 (AFB1) antibodies (Abs), cross-reacting with other aflatoxins, which were able to hinder, proportionally to their titer, the secretion of aflatoxin M1 (AFM1) into the milk of cows continuously fed with AFB1. According to anti-AFB1 Ab titer, 50% of the vaccinated cows were recognized as high responder animals. In an attempt to prepare a more effective formulation for vaccination of cows, it was compared the immunogenicity, in Holstein Friesian heifers, of AnAFB1 covalently conjugated to KLH or to recombinant diphtheria toxin (CRM197) molecules, and injected together with various adjuvants. This study demonstrated that injection of AnAFB1 conjugated to KLH and mixed with complete (priming) and incomplete Freund's adjuvant (boosters), as in the previous schedule of immunization, was the most effective regimen for inducing Ab responses against AFB1, although pre-calving administration could increase the effectiveness of vaccination, resulting in 100% high responder animals. After one booster dose at the beginning of the milk production cycle, anti-AFB1 Ab titers were comparable to those recorded at the end of the immunization schedule, and proved to be effective in reducing significantly AFB1 carry over, as AFM1, from feed to milk. Pre-calving vaccination of dairy heifers with conjugated AnAFB1, adjuvated with complete and incomplete Freund's adjuvant, may represent the most effective tool for preventing the public health hazard constituted by milk and cheese contaminated with aflatoxins.