Resistin is a cytokine implicated in the development of insulin resistance. However, there has been little investigation of the effects of resistin on FA metabolism and insulin response in skeletal muscle, a key tissue for glucose disposal. The purpose of the present study was to examine the role of altered fatty acid (FA) metabolism as a cause of resistin's inhibition of insulin-stimulated glucose transport in muscle. Isolated rat soleus muscles were incubated acutely (2 hrs) in the presence or absence of 600 ng/mL resistin, with or without 2 mM palmitate. Resistin acutely impaired insulin-stimulated glucose transport and Akt phosphorylation, but only in the presence of palmitate, implicating a role for altered FA metabolism. This impairment of glucose transport induced by resistin plus palmitate could be pharmacologically rescued by the inclusion of AICAR, a stimulator of AMP-activated protein kinase and FA oxidation, as well as inhibitors of ceramide synthesis (myriocin, fumonisin). However, to our surprise, resistin actually blunted the palmitate-induced increase in muscle ceramide content; as expected, ceramide content was significantly lowered by fumonisin. In summary, the acute impairment of insulin response by resistin was manifested only in the presence of high palmitate, and was alleviated when FA metabolism was manipulated (increased oxidation, inhibited ceramide synthesis). Resistin's acute impairment of insulin response does not appear to require an absolute increase in ceramide content; however reducing ceramide content alleviated the impairment in glucose transport and insulin signaling.