Abstract: This study aims to investigate the protective effects of the extract of Sophora japonica L. (Sophora extract) and vitamin C (V_C) on UV-induced damage in HaCaT cells. HaCaT cells were cultured in vitro and divided into different groups: Control group (without UV irradiation or treatment with Sophora extract, V_C, or their combination), UV radiation group, 125 μg/mL Sophora extract group (with UV irradiation and treatment with Sophora extract), 125 μg/mL vitamin C group (with UV irradiation and treatment with V_C), and 125 μg/mL combination group (with UV irradiation, Sophora extract and V_C at a mass ratio of 1:1). The inhibitory effects of Sophora extract, V_C, and their combination on reactive oxygen species (ROS) production and mitochondrial ROS in UV-irradiated HaCaT cells were measured using fluorescence staining and enzyme-linked immunosorbent assay. Immunofluorescence staining and enzyme-linked immunosorbent assay were also employed to evaluate the expression levels of cyclobutane pyrimidine dimers (CPDs) and the inflammatory cytokine IL-6 in UV-irradiated HaCaT cells treated with Sophora extract, V_C, or their combination. The results showed that the total ROS inhibition rates of Sophora extract, V_C, and the combination in UVA-treated HaCaT cells were 38.23%±9.23%, 27.20%±6.87%, and 64.13%±4.08%, respectively. The combination significantly enhanced the total ROS inhibition rate compared to the individual treatments (P<0.01). The inhibition rates of mitochondrial ROS (mtROS) by Sophora extract, V_C, and the combination were 50.29%±4.92%, 38.81%±8.66%, and 84.74%±3.68%, respectively. The combination significantly increased the mtROS inhibition rate compared to the individual treatments (P<0.01). After UVB irradiation, the CPDs content in HaCaT cells increased, and Sophora extract, V_C, and their combination significantly reduced CPDs formation (P<0.01), with the combination showing a more pronounced effect compared to the Sophora extract group (P<0.05) and the V_C group (P<0.01) alone. Furthermore, Sophora extract and the combination significantly reduced the secretion of the inflammatory cytokine IL-6 in HaCaT cells after UVA irradiation (P<0.01, P<0.05), indicating an anti-inflammatory capability. The results demonstrated that the combination of Sophora extract and Vitamin C could alleviate oxidative stress damage and inflammatory responses caused by UV radiation and provided experimental support for future in vivo studies and the development and utilization of health-promoting Sophora extract and V_C combinations.