: Compared with trivalent influenza vaccines, quadrivalent influenza vaccines are expected to provide wider protection against influenza B virus infections. We developed a novel quadrivalent subunit influenza vaccine which was distinct from the influenza vaccines available on the market in production process. In this research, we evaluated the safety and immunogenicity of the quadrivalent subunit influenza vaccine in animal models.: In toxicity assessment, 40 SD rats were randomly assigned to be intramuscularly injected with 1.0 ml of the tested vaccine (33 μg/ml) or 0.9% sodium chloride solution. In irritation assessment, eight rabbits were randomly assigned to receive 0.5 ml of tested vaccine or phosphate buffer solution intramuscularly. Thirty-two guinea pigs were randomly assigned to be intramuscularly injected with high-dose tested vaccine (0.5 ml), low-dose tested vaccine (0.05 ml), ovalbumin, or 0.9% sodium chloride solution, respectively, for sensitization assessment. In immunogenicity assessment, 50 BALB/c mice were equally randomized to receive one dose of tested vaccine, two doses of tested vaccine with an interval of 14 days, 0.5 ml of trivalent subunit influenza vaccine, 0.5 ml of monovalent subunit influenza vaccine, or 0.5 ml of phosphate buffer solution. Orbital blood was collected before and 28 and 42 days after administration of the injections for detecting influenza antibody titers.: No abnormal toxicity and irritation in rats and rabbits showed in the gross autopsy and histopathological examinations. The results of sensitization in guinea pigs indicated that no obvious allergic symptoms observed in the high-dose and low-dose vaccine groups within 30 min after twice provocations, and the result of sensitization evaluation was negative. Vaccine induced significant immune responses in mice with 100% seroconversion rates at 28 and 42 days after the first dose. The geometric mean titers (GMTs) of hemagglutination inhibition (HI) antibodies at day 28 in one-dose quadri-vaccine and two-dose quadri-vaccine groups were comparable to those in the tri-vaccine or mono-vaccine groups for shared influenza strains. However, the GMTs of HI antibodies against H1N1 ( = 0.025) and BV ( = 0.049) at day 42 in one-dose quadri-vaccine group were significantly lower than those in the tri-vaccine or mono-vaccine groups. The GMTs of HI antibodies against H1N1, H3N1, BY, and BV at day 28 and day 42 were comparable between one-dose quadri-vaccine and two-dose quadri-vaccine groups.: The quadrivalent subunit influenza vaccine was safe and immunogenic in animal models. One dose of the vaccine could elicit a satisfactory antibody response in mice.