Hypoxia-inducible factor-1α (HIF-1α) is considered the main transcriptional regulator of the hypoxia-specific cellular and developmental response. This study was performed to investigate the effect of Shenqin biochemical extract (SQBE) on HIF-1α expression in ultraviolet B (UVB)-irradiated HaCaT cells and the possible action mechanisms of SQBE against UVB-induced skin cancer. HaCaT cells in logarithmic growth phase were seeded in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, and conventionally cultured at 37°C with 5% CO2. Cells were divided into control group (administered the same amounts of dimethyl sulfoxide), SQBE1 group (12.5 μg/mL SQBE), SQBE2 group (25.0 μg/mL SQBE), and SQBE3 group (50.0 μg/mL SQBE). Four hours post administration, the control and treatment groups were irradiated with UVB (0, 20, 40, and 60 mJ/cm2). After 24 h, cell survival rate was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression levels of HIF-1α mRNA and protein were detected by polymerase chain reaction and western blotting, respectively. SQBE-treated, UVB-irradiated cells had improved survival rates. This increase was most significant in SQBE3 group (P < 0.01), which also had effectively reduced expression of intracellular HIF-1α mRNA and protein. Hence, SQBE had a protective effect on UVB-irradiated HaCaT cells and inhibited the UVB irradiation-induced expression of HIF-1α. This indicates that SQBE could prevent the occurrence of UVB radiation-induced skin cancer.