Objective To confirm the performance of the pulsating vacuum sterilizer and its effect on SPF golden hamster cages and drinking water under specified loading and sterilization conditions. Methods The pulsating vacuum sterilization of different articles in the same load were performed by chemical and physical method with a professional temperature probe and biological indicator. First, a no-load pressure holding test and B-D test of the pulsating vacuum sterilizer and no-load heat distribution test were performed to confirm the performance of the sterilizer itself. Then, full-load heat distribution / heat penetration / microbial challenge, and asepsis tests were performed according to the specified loading method and sterilization conditions. Results The performance of the pulsating vacuum sterilizer was determined: vacuum holding pressure, B-D test, and no-load heat distribution test were all qualified. The test result of SPF golden hamster cages and drinking water subjected to full-load heat distribution / heat penetration / microbial challenge tests in accordance with specified loading method and sterilization conditions were qualified. After high pressure conditions, the sampling pads and drinking water were placed in culture medium for 14 days, but no bacterial colony growth was observed indicating the success of the sterility test. Conclusions SPF golden hamster cages and drinking water can be sterilized by pulsating vacuum sterilization under specified loading method and sterilization conditions.