Gonadotrophin-releasing hormone (GnRH) is the prime decapeptide hormone in the regulation of mammalian reproduction. Active immunization against GnRH has been a good treatment option to fight against hormone-dependent disease such as breast cancer. We designed and purified a novel protein vaccine Hsp65–GnRH6 containing heat shock protein 65 (Hsp65) and six copies of GnRH in linear alignment. Immunization with Hsp65–GnRH6 evoked strong humoral response in female mice. The generation of specific anti-GnRH antibodies was detected by ELISA and verified by western blot. In addition, anti-GnRH antibodies effectively neutralized endogenous GnRH activity in vivo, as demonstrated by the degeneration of the ovaries and uteri in the vaccinated mice. Moreover, the growth of EMT-6 mammary tumor allografts was inhibited by anti-GnRH antibodies. Histological examinations have shown that there was increased focal necrosis in tumors. Taken together, our results showed that immunization with Hsp65–GnRH6 elicited high titer of specific anti-GnRH antibodies and further led to atrophy of reproductive organs. The specific antibodies could inhibit the growth of EMT-6 murine mammary tumor probably via an indirect mechanism that includes the depletion of estrogen. In view of these results, the protein vaccine Hsp65–GnRH6 appears to be a promising candidate vaccine for hormone-dependent cancer therapy.