[Objective] To define and compare the parameters for the heat and formaldehyde inactivation of Brucella strains, and provide information for preparation of Brucella inactivated antigen. [Methods] The four Brucella virulent reference strains 2308 (B. abortus), M28 (B. melitensis), S1330 (B. suis) and 16M (B. melitensis) were cultivated in TSA media, and the harvested culture were resuspended to a concentration of (4?8)×1010 CFU/mL by saline. Then the suspension was divided into the equal two parts for heat inactivation at 80 oC for different time or formaldehyde inactivation at different concentration and for different time at 37 oC, and the inactivation effects for the two methods were compared. The rabbits of 1.5?2.0 kg were subcutaneously inoculated with the heat or formaldehyde inactivated 16M antigen at the dosage of 1×1010 CFU/mL. The serum antibody titer was measured weekly during six weeks post inoculation by Rose Bengal test (RBT) and Serum agglutination test (SAT). [Results] It takes 5 min to inactivate Brucella strains 2308, S1330 and 16M by heating at 80 oC, and 10 min for all four strains. The four Brucella strains could not be completely inactivated while incubated in 0.2% formaldehyde even for 7 days. The strains 16M and M28 could be inactivated thoroughly via incubation in 0.4% formaldehyde for 12 or 72 hours, respectively. Although the effect varied for the inactivation of strains 2308 and S1330 in two independent experiments, the four strains could be entirely inactivated by incubation in 0.6% formaldehyde for 72 hours. The fluctuation trends were almost consistent for the stimulated serum antibodies against the two heat or formaldehyde inactivated 16M antigens. Moreover, the antibody titer against the formaldehyde inactivated antigen was slightly better than that against the heat inactivated antigen. [Conclusion] Heat inactivation at 80 oC and inactivation in 0.6% formaldehyde could be used to prepare Brucella inactivated antigen and did not change the immunogenicity of Brucella.