Gastric cancer (GC) is a highly prevalent and aggressive malignancy with a poor prognosis. Recent evidence suggested that metallothionein 1 M (MT1M) may play a critical role in cancer development, progression, and drug resistance; however, its role in GC remains largely unknown. In this study, we investigated the expression and function of MT1M in GC both in vitro and in vivo. We found that MT1M expression was significantly downregulated in GC tissues and cell lines. Decreased expression of MT1M was associated with worse clinical prognosis, particularly in patients treated with 5-fluorouracil. Low expression of MT1M was indicative of poor overall survival (OS, HR 0.56 [95% CI 0.37–0.84], P < 0.005), first progression survival (FP, HR 0.54 [95% CI 0.36–0.79], P < 0.005), and post-progression survival (PPS, HR 0.65 [95% CI 0.45–0.94], P < 0.05). We also demonstrated that overexpression of MT1M inhibited cell proliferation and induced apoptosis in GC cells and in tumor xenografts, and it improved chemosensitivity to 5-fluorouracil. Furthermore, we found that MT1M overexpression could inhibit stem cell characteristics by targeting GLI1 and affecting GLI1 ubiquitination. Collectively, these findings indicated that MT1M may act as a tumor suppressor in GC and could serve as a potential therapeutic target to attenuate stemness and chemotherapy resistance of GC. [Display omitted] • MT1M is downregulated in GC tissues and is associated with poor prognosis. • Overexpression of MT1M inhibits malignant phenotypes in GC cells. • MT1M may suppress GC progression and stemness by inhibiting GLI1. • MT1M overexpression could be a potential therapeutic target for GC. [ABSTRACT FROM AUTHOR]