BackgroundHypoxia-induced glycogen turnover is implicated in cancer proliferation and therapy resistance. Triple-negative breast cancers (TNBCs), characterized by a hypoxic tumor microenvironment, respond poorly to therapy. We studied the expression of glycogen synthase 1 (GYS1), the key regulator of glycogenesis, and other glycogen-related enzymes in primary tumors of patients with breast cancer and evaluated the impact ofGYS1downregulation in preclinical models.MethodsmRNA expression ofGYS1and other glycogen-related enzymes in primary breast tumors and the correlation with patient survival were studied in the METABRIC dataset (n= 1904). Immunohistochemical staining of GYS1 and glycogen was performed on a tissue microarray of primary breast cancers (n= 337). In four breast cancer cell lines and a mouse xenograft model of triple-negative breast cancer,GYS1was downregulated using small-interfering or stably expressed short-hairpin RNAs to study the effect of downregulation on breast cancer cell proliferation, glycogen content and sensitivity to various metabolically targeted drugs.ResultsHighGYS1mRNA expression was associated with poor patient overall survival (HR 1.20,P= 0.009), especially in the TNBC subgroup (HR 1.52,P= 0.014). Immunohistochemical GYS1 expression in primary breast tumors was highest in TNBCs (median H-score 80, IQR 53–121) and other Ki67-high tumors (median H-score 85, IQR 57–124) (P< 0.0001). Knockdown ofGYS1impaired proliferation of breast cancer cells, depleted glycogen stores and delayed growth of MDA-MB-231 xenografts. Knockdown ofGYS1made breast cancer cells more vulnerable to inhibition of mitochondrial proteostasis.ConclusionsOur findings highlight GYS1 as potential therapeutic target in breast cancer, especially in TNBC and other highly proliferative subsets.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-023-02715-z.