The saturated fatty acid (SFA) palmitate (C16:0) induces insulin resistance in cultured skeletal muscle cells, which may be related to NF-B activation. The aim of this study was to evaluate if other SFA also exert these effects on skeletal muscle and whether these relate to chain length. Therefore, we incubated L6 and C2C12 skeletal muscle cells with four different fatty acids: caprylate (C8:0), laurate (C12:0), palmitate (C16:0) and stearate (C18:0) to study effects on GLUT4 translocation, deoxyglucose uptake and NF-B activation. Incubation of L6 cells with the long-chain FA C16:0 and C18:0 resulted in a reduced insulin-stimulated GLUT4 translocation and deoxyglucose uptake, while L6 cells incubated with the medium-chain FA C8:0 and C12:0 remained insulin sensitive. Besides increasing NF-B DNA binding activity in both L6 and C2C12 cells, C16:0 also induced NF-B transcriptional activity. C18:0 showed comparable effects, while the SFA with shorter chain lengths were not able to elevate NF-B transcriptional activity. Collectively, these results demonstrate that SFA-induced NF-B activation coincides with insulin resistance and depends on FA chain length.