Type 2-diabetes is a significant and rapidly growing health problem. Skeletal muscle is the major site of insulin-mediated glucose uptake and this process is impaired in type 2-diabetes. The signalling cascade leading to glucose uptake by skeletal muscles is not fully understood; for instance, the involvement of Ca²⁺ is a matter of debate. Here we examine the role of Ca²⁺ influx in insulin-mediated glucose uptake in skeletal muscle, specifically focusing on the possible involvement of canonical transient receptor potential (TRPC) channels. The study was approved by the local ethics committee. Mice were killed by rapid cervical dislocation. Ca²⁺ influx was assessed in isolated single muscle cells by measuring the Ba²⁺ influx with the fluorescent indicator Calcium Green-1 and confocal microscopy. Glucose uptake was assessed by measuring the uptake of radioactively labelled 2-deoxyglucose in isolated extensor digitorum longus (EDL) and soleus muscles. The results show an insulin-mediated increase in Ba²⁺ influx (16 ± 2%; mean ± SEM, n = 34; P < 0.001, paired t-test), which was blocked by the TRP channel inhibitors 2-aminoethoxydiphenyl borate (2-APB, 100 μM) and Gd3+ (1 μM). 2-APB also decreased insulin-mediated glucose uptake in EDL and soleus muscles in a dose-dependent manner (~50% inhibition at 100 μM). 2-APB had no effect on basal, contraction- or hypoxia-mediated glucose uptake. The TRPC3/6/7 subfamily is known to be activated by diacylglycerol (DAG) and we observed an increased Ba²⁺ influx in response to the membrane permeable DAG analogue 1-oleyl-2-acetyl-sn-glycerol (OAG, 30 μM), which was blocked by 2-APB and Gd³⁺. When OAG was added in the continued presence of insulin, there was an additional ~30% increase in the Ba²⁺ influx as compared to insulin alone (P < 0.001, paired t test, n = 14). In the presence of insulin plus OAG, glucose uptake in EDL muscles was increased by ~25% compared to insulin alone (P < 0.01, unpaired t test, n = 8). OAG had no effect on basal glucose uptake. In conclusion, Ca²⁺ influx appears to have a specific role in insulin-mediated glucose uptake. Our results indicate that TRPC channels are involved in this Ca²⁺ signalling and hence these channels provide a novel target for therapeutic interventions in insulin resistant conditions.