Betulinic acid (BA) exhibits various biological activities such as anti-bacterial, anti-inflammatory, antihuman papilloma virus (HPV), and anti-cancer activities. HPV infection is associated with a high risk of cervical cancer, which is the leading cause of deaths among women worldwide. Therefore, BA is an attractive therapeutic agent for treating cervical cancer. In this study, we investigated the role of BA in regulating the hypoxia-mediated response in HeLa cells and clarified the underlying mechanism of action. We found that BA inhibited the hypoxia-induced accumulation of HIF-1 alpha without affecting HIF-1 alpha mRNA levels and suppressed the expression of HIF target genes, including VEGF, GLUT1, and PDK1 in HeLa cells. Additionally, BA enhanced the 131,132, and 135 activities of the proteasome, which resulted in reduced levels of ubiquitinated proteins and HIF-1 alpha protein in HeLa cells. However, BA treatment did not affect the deubiquitinase enzyme activity in HeLa cells. These results indicate that inhibition of HIF-1 alpha accumulation by BA is mediated by activation of the proteasome, and BA is a potential anticancer agent for the regulation of the HIF signaling pathway. (C) 2020 Elsevier Inc. All rights reserved.