The molecular mechanisms of obesity-associated insulin resistance are becoming increasingly clear, and the effects of various lipid molecules, such as diacylglycerol and ceramide, on the insulin signal are being actively explored. To better understand the divergent response to lipid exposure between lean and obese, we incubated primary human muscle cells from lean [body mass index (BMI) <25 kg/m²] and morbidly obese (BMI >40 kg/m²) subjects with the saturated fatty acid palmitate. Additionally, given that AMPK-activating drugs are widely prescribed for their insulin-sensitizing effects, we sought to determine whether 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR)-stimulated AMPK activation could prevent or reverse the deleterious effects of lipid on insulin signaling. We found that a 1-h palmitate incubation in lean myotubes reduced (P < 0.05) insulin-stimulated phosphoprotein kinase B (Akt), Akt substrate 160 (AS160), and inhibitory factor κBα (IκBα) mass, all of which were prevented with AICAR inclusion. With a longer incubation, we observed that myotubes from morbidly obese individuals appear to be largely resistant to the detrimental effects of 16 h lipid exposure as was evident, in contrast to the lean, by the absence of a reduction in insulin-stimulated insulin receptor substrate (IRS)-1 Tyr phosphorylation, phospho-Akt, and phospho-AS160 (P < 0.05). Furthermore, 16 h lipid exposure significantly reduced IκBα levels and increased phosphorylation of c-Jun NH₂-terminal kinase (JNK) and IRS1-Ser³¹² in lean myotubes only (P < 0.05). Despite a divergent response to lipid between lean and obese myotubes, AICAR inclusion improved insulin signaling in all myotubes. These findings suggest an important role for regular exercise in addition to offering a potential mechanism of action for oral AMPK-activating agents, such as thiazolidinediones and metformin.