The hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxic stress under physiological and pathological conditions. HIF-1α protein stability is tightly regulated by the ubiquitin-proteasome system (UPS) and autophagy in normoxia, hypoxia, and the tumor environment to mediate the hypoxic response. However, the mechanisms of how the UPS and autophagy interplay for HIF-1α proteostasis remain unclear. Here, we found a HIF-1α species propionylated at lysine (K) 709 by p300/CREB binding protein (CBP). HIF-1α stability and the choice of degradation pathway were affected by HIF-1α propionylation. K709-propionylation prevented HIF-1α from degradation through the UPS, while activated chaperon-mediated autophagy (CMA) induced the degradation of propionylated and nonpropionylated HIF-1α. CMA contributed to HIF-1α degradation in both normoxia and hypoxia. Furthermore, the pan-cancer analysis showed that CMA had a significant positive correlation with the hypoxic signatures, whereas SIRT1, responsible for K709-depropionylation correlated negatively with them. Altogether, our results revealed a novel mechanism of HIF-1α distribution into two different degradation pathways. [BMB Reports 2023; 56(4): 252-257].