One of the primary characteristics of tumour cells is the reliance on glucose as a fuel source. We have shown that the central regulatory protein of the specialised actin cytoskeleton found in tumour cells, the tropomyosin Tpm3.1, regulates glucose uptake (Kee et al. Traffic 16: 691-711, 2015). We have taken a novel approach, and designed compounds that target this Tpm and shown they have anti-proliferative effects and little cardiotoxicity (Stehn et al. Can Res 16: 5169-82, 2013). The aim of the present study is to define the on- and off-target impacts of anti-Tpm (ATM) drugs on glucose metabolism using wild-type (WT) and Tpm3.1 knock-out (KO) mice. Both chronic (7 days) and acute (1h) treatment with the ATMs drugs, TR100 and ATM1001 (40mg/kg body weight/day), led to raised blood glucose and insulin levels, suggesting an impact of the drug on insulin sensitivity. TR100 and ATM1001-treated mice had poorer glucose clearance due mainly to suppression of glucose-stimulated insulin secretion from pancreatic islets. In addition, TR100 and ATM1001 suppressed basal (20%) insulin-stimulated glucose uptake (75%) in skeletal muscle ex vivo. Importantly, the impact of the drug (glucose clearance, insulin secretion and glucose uptake) was considerably less in the KO vs WT mice (P < 0.05; 2-way ANOVA) indicating that the ATM drug affect glucose metabolism largely by inhibiting Tpm3.1's function. In conclusion, we have demonstrated that the ATM drugs targeting Tpm3.1 reduce glucose metabolism in the whole animal which in turn will contribute to the anticancer activity of these first-in-class drugs. These studies establish tropomyosin as a novel target to reduce both glucose metabolism and cell proliferation of cancer cells.