Background <p>Glyoxalase 1 (GLO1), an enzyme responsible for breaking down methylglyoxal (MG), is increasingly recognized as a tumor-promoting factor due to its role in removing cytotoxic MG. This study aims to analyze the expression status and prognostic significance of GLO1 across various cancers and explore its potential role in cancer immunotherapy.</p> Methods <p>We obtained GLO1 mRNA expression profiles from The Cancer Genome Atlas (TCGA) database. Kaplan-Meier analysis was used to evaluate the prognostic value of GLO1 in multiple cancers. We validated GLO1 protein expression in clinical endometrial cancer samples using immunohistochemical staining. Additionally, we analyzed GLO1’s correlation with drug sensitivity from the Genomics of Drug Sensitivity in Cancer (GDSC) database and its relationship with the tumor microenvironment and immunotherapy response.</p> Results <p>GLO1 was widely expressed in human tumor tissues with significant differences compared to adjacent normal tissues. High GLO1 expression was associated with poor prognosis in 14 cancers, including adrenocortical carcinoma, cholangiocarcinoma, and lymphoid neoplasm diffuse large B-cell lymphoma. GLO1 expression was negatively related to immune cell infiltration in breast invasive carcinoma. In melanoma and kidney renal clear cell carcinoma, high GLO1 expression was linked to poor immunotherapy outcomes.</p> Conclusions <p>This comprehensive pan-cancer study reveals the prognostic value and potential predictive role of GLO1 in immunotherapy across multiple tumor types. This study primarily utilized bioinformatic datasets; experimental validation was performed for endometrial cancer but warrants expansion to other cancer types in future work.</p>