ABSTRACT Gastrointestinal stromal tumor (GIST) frequently develops resistance to imatinib (IM). This study identifies AOC3 downregulation as a critical contributor to IM resistance. Analyzing clinical samples and IM‐sensitive/resistant GIST cell lines, we found AOC3 significantly decreased in resistant states. Functionally, AOC3 knockdown promoted IM resistance, enhanced glycolytic activity, and increased lactate production. Mechanistically, AOC3 loss attenuated ubiquitin‐mediated degradation of HK2, stabilizing this glycolytic enzyme and boosting lactate generation. Subsequent histone lactylation, notably at H3K18, enriched at the Myc promoter and stimulated its transcription. Crucially, HK2 overexpression reversed AOC3's suppressive effects on glycolysis, lactylation, Myc expression, and IM resistance. In vivo xenograft models confirmed these findings. We conclude that AOC3 acts as a tumor suppressor by promoting HK2 degradation; its loss triggers a HK2/glycolysis/lactylation/Myc axis driving IM resistance, positioning AOC3 as a promising prognostic biomarker and therapeutic target in GIST.