Uterine contraction is crucial for a successful labor and the prevention of postpartum hemorrhage. It is enhanced by hypoxia; however, its underlying mechanisms are yet to be elucidated. In this study, transcriptomes revealed that hypoxia-inducible factor (HIF)-1α was up-regulated in laboring myometrial biopsies, while blockade of HIF-1α decreased the contractility of the myometrium and myocytes in vitro via siRNA and the inhibitor, 2-methoxyestradiol. Chromatin immunoprecipitation sequencing revealed that HIF-1α directly binds to the genome of contraction-associated proteins: the promoter of GJA1 and PTGS2, and the intron of OXTR. Silencing the HIF-1α reduced the expression of PTGS2, GJA1, and OXTR. Furthermore, blockade of GJA1 or PTGS2 led to a significant decrease in myometrial contractions in the hypoxic tissue model, whereas atosiban did not remarkably influence contractility. Our study demonstrates that HIF-1α is essential for promoting myometrial contractility under hypoxia by directly targeting GJA1 and PTGS2, but not OXTR. These findings help us to better understand the regulation of myometrial contractions under hypoxia and provide a promising strategy for labor management and postpartum hemorrhage treatment.