We developed a coculture system comprising primary rat adipocytes and L6 rat skeletal muscle cells to allow investigation of the effects of physiologically relevant mixtures of adipokines. We observed that coculture, or adipocyte-conditioned media, increased glucose uptake in muscle cells. An adipokine which could potentially mediate this effect is adiponectin and we demonstrated that siRNA-mediated knockdown of adiponectin receptor (adipoR)-2 in muscle cells reduced the uptake of glucose upon coculture with primary rat adipocytes. Analysis of coculture media by ELISA indicated total adiponectin concentration of up to 1 µg/ml and Western blotting and gel filtration analysis demonstrated that the adipokine profile was hexamer > HMW >> trimer. We used the streptozotocin-induced rat model of diabetes and found that HMW adiponectin levels decreased in comparison with control animals and this correlated with the fact that STZ rat-derived primary adipocytes in coculture did not stimulate glucose uptake to the same extent as control adipocytes. Coculture induced phosphorylation of AMPK (T172) and interestingly also IRS1 (Y612) and Akt (T308 & S473) which could be attenuated after AdipoR2-siRNA treatment. In summary, we believe that this coculture system represents an excellent model to study the effects of primary adipocyte-derived adipokine mixtures on skeletal muscle metabolism and here we have established that in the context of physiologically-relevant mixtures of adipokines, adiponectin may be an important determinant of positive crosstalk between adipocytes and skeletal muscle.