Hypoxia-inducible factor-1-alpha (HIF-1α) activates the transcription of many genes that code for proteins involved in angiogenesis, glucose metabolism, cell proliferation/survival, and invasion/metastasis. However, the mechanisms between HIF-1 and its downstream target genes are still poorly understood. Our experimental results had shown that nuclear HIF-1α proteins were significantly induced after HepG2 cells treatment with 1% O₂ for 6 h and reached the peak expression level in 24 h. Meanwhile, the results of RT-qPCR and Western-blotting showed that HIF-1α and cathepsin B (CTSB) expressions increased with a similar pattern in response to hypoxia in the HepG2 cells. Additionally, based on bioinformatics analysis, we identified a hypoxia response element in the CTSB promoter, indicating a possible association between HIF-1α and CTSB. Moreover, luciferase assay was performed to reflect the transcriptional activity mediated through the HIF-1α binding HRE within the CTSB promoter. Furthermore, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (CHIP) revealed a specific HIF-1α binding activity to the CTSB gene promoter. For the first time, we demonstrated that CTSB is a new HIF-1α-target gene. We believe these findings will contribute to the research and development of neoplasm-targeted therapies.