Studies have shown the implication of specific adipokines or fatty acids (FA) in the pathogenesis of insulin resistance. However, the interplay of adipokines with FA remains poorly understood. This study aimed to investigate the combined effects of adipokines and low concentrations of palmitic acid (PA, 100 µmol/l) on skeletal muscle metabolism. Human skeletal muscle cells were incubated with adipocyte-conditioned medium (CM), PA or PA+CM, and FA transporter and FA metabolism were analysed. CM-incubation increased CD36 level (1.8 fold) and PA-uptake (1.4 fold). However, only co-application of PA+CM resulted in profound lipid accumulation (5.3 fold), 60% reduction of PA-oxidation and 3.5 fold increased diacylglycerol content. Our results support a novel role for adipokines in the pathogenesis of T2D by increasing the lipotoxic potential of PA, notably of low concentrations. This implies an increased lipotoxic risk already at an early stage of weight gain, when lipolysis has not yet contributed to increased plasma free FA levels.