Abstract: This study aims to evaluate the immunogenicity and safety of the 20-valent pneumococcal conjugate vaccine (PCV20). Different evaluation models were established using ICR mice and SD rats to assess the immunogenicity and toxic reactions. The results showed that the immunogenicity test in mice demonstrated good immunogenicity for three batches of the 20-valent pneumococcal conjugate vaccine after three doses. The immunogenicity test in rats showed that the vaccine induced antibodies against 20 pneumococcal serotypes within 2 weeks after the first dose and maintained a certain level for 3 weeks after the final dose, indicating good immune persistence. The vaccine was administered to mice at the maximum dosage without any significant toxic reactions observed. The repeated-dose safety test in rats showed local irritation and pathological changes at the injection site, which were alleviated during the recovery period. No significant toxic reactions were observed at the end of the dosing period and the recovery period. Studies have shown that the novel 20-valent pneumococcal conjugate vaccine exhibits good immunogenicity and safety in mouse and rat models. The use of all experimental animals has been approved by the Ethics Committee of Laboratory Animal of Beijing Minhai Biological Technology Co. Ltd. (No. 202309, 202311).