BACKGROUND: The aetiology and pathogenesis of adenomyosis remain unclear. This study utilised untargeted metabolomics to explore the aberrant amino acid metabolism in adenomyosis. Among the studied metabolites, hydroxyproline is known to promote the proliferation and invasion of tumour cells. Due to detection limitations, hydroxyproline levels were inferred METHODS: Endometrial metabolomic analysis was performed using gas chromatography-mass spectrometry (GC-MS) in 15 and 20 patients with and without adenomyosis. The expression levels of P4HA1 mRNA and protein were detected using quantitative PCR (qPCR), western blotting, and immunohistochemistry. RESULTS: In total, 22 differentially expressed metabolites were identified in the adenomyosis group compared to the control group. qPCR results demonstrated that P4HA1 mRNA expression levels were significantly higher in the adenomyosis group than in the control group ( CONCLUSION: Hydroxyproline levels were significantly elevated in the endometrial tissues of patients with adenomyosis. Furthermore, hydroxyproline promotes the proliferation and migration of hESCs. These findings provide new insights into the pathogenesis of adenomyosis and suggest potential therapeutic strategies.