Objective To establish tumor necrosis factor receptor superfamily member 4 (TNFRSF4) knockout and humanized mice for TNFRSF4-targeted cancer therapeutic antibodies development, and to study the function of TNFRSF4 in the immune response. Methods The TNFRSF4 knockout and humanized mice were established with the CRISPR/ Cas9 method. The mice were characterized by PCR, RT-PCR, HE staining and fluorescence-activated cell sorting. Results We successfully generated mice expressing the human TNFRSF4 gene, as well as TNFRSF4 gene knockout mice. TNFRSF4 humanized mice expressed human TNFRSF4, but not murine TNFRSF4. No TNFRSF4 expression was detected in TNFRSF4 knockout mice. TNFRSF4 humanized mice and knockout mice survived normally within 6 months after birth, and there were no obvious abnormalities in histopathology and immune system. Conclusions TNFRSF4 humanized mice and knockout mice produced using CRISPR/ Cas9 technology can be used as animal models for screening and evaluating of TNFRSF4-targeted cancer therapeutic antibodies or for studying the role of TNFRSF4 in immune processes.