Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in type 2 diabetes and insulin resistance. In a previous study, we reported that mixed D, L isomers of C12- or C14-carnitine induced an NFB-luciferase reporter gene in RAW 264.7 cells suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical pro-inflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of COX-2 in a chain length dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25μM), used as a representative acylcarnitine, stimulated the expression and secretion of pro-inflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many pro-inflammatory signaling pathways including PRRs. Knockdown of MyD88, a key co-factor in PRR signaling and inflammation, blunted the pro-inflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking TLR2 and TLR4, and did not activate reporter constructs in TLR-overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.