Background: High-intensity focused ultrasound (HIFU) represents a therapeutic medical procedure that operates by inducing ablation and mechanical disruption. Despite its established efficacy, its potential impact on tumor chemotherapy remains uncertain. Long noncoding RNA (lncRNA) MAFA-AS1 facilitated cancer cell proliferation and fostering drug resistance, but the precise significance and functional implications of MAFA-AS1 in the context of ovarian cancer (OC) remain largely unexplored. The objective of this experiment is to further investigate the potential of HIFU in inhibiting the chemotherapy resistance mechanism of OC. Materials and Methods: Five types of human ovarian cancer cells were employed in this study. There were four different groups, namely MAFA-AS1 siRNA, si-NC, pcDNA3.1-HIF-1 (hypoxiainducible factor-1), and pcDNA3.1-control. Quantitative real-time PCR, cell proliferation assay, apoptosis assay, western blot assay, subcellular fractionation, aerobic glycolysis analysis, RNA immunoprecipitation (RIP), and luciferase reporter assay were conducted for experimentation and validation. Results: Significant upregulation of MAFA-AS1 in OC cells was observed. Through loss-of-function experiments based on HIFU, we unraveled its oncogenic functions in OC. MAFA-AS1 was discovered to bind to HIF-1 mRNA, thereby enhancing its expressed stability. Further investigations revealed an interaction between MAFA-AS1 and fat mass and obesity-associated protein (FTO), positively modulating HIF-1 mRNA stability in an FTO -dependent manner. Importantly, MAFA-AS1 exerts its effects on OC by acting through HIF-1. Conclusion: The study underscores the role of MAFA-AS1 in promoting aerobic glycolysis and chemical resistance in OC by up-regulating HIF-1 expression, suggesting that targeting MAFA-AS1 holds promise as a therapeutic strategy for OC patients undergoing chemotherapy.