The Escherichia coli K88, K99, 987P strains and C type Clostridium perfringens were used to prepare piglets colibacillosis. Pregnant sows were immunized with different doses of vaccine, and then their piglets were challenged with the corresponding pathogenic strains and doses. The results showed that the protection rate of the piglets was above 80% when the vaccine doses was 1.6 mL or more. The protection rate of piglets against bacteria was not less than 80% when piglets aged 1, 4,7 and 10 d were challenged, which indicated that the immunization period was within 24 h after colostrum feeding and the duration of passive immunization was more than 7 d. Three batches of vaccines were kept at 28℃ and tested at different times during preservation. The results showed that the vaccine was safe for pregnant sows after 27 months of preservation and the protective rate of piglets produced by the vaccine immunized sows was no less than 80%. Therefore, the minimum immune dose of the vaccine was 1.6 mL, the passive immunization duration of piglets was more than 7 d, and the vaccine storage period was 24 months at 28℃.