Among dietary components, conjugated linoleic acids (CLAs) have attracted considerable attention as weight loss supplements in the Western world because they reduce fat stores and increase muscle mass. However, a number of adverse effects are also ascribed to the intake of CLAs such as aggravation of insulin resistance and the risk of developing diabetes. However, the mechanisms accounting for the effects of CLAs on glucose homeostasis are incompletely understood. Herein we provide evidence that CLAs specifically activate the cell surface receptor FFA1, an emerging therapeutic target to treat type 2 diabetes. Using different recombinant cellular systems engineered to stably express FFA1 and a set of diverse functional assays including the novel, label-free non-invasive dynamic mass redistribution technology (Corning® Epic® biosensor), both CLA isomers cis-9, trans-11-CLA and trans-10, cis-12-CLA were found to activate FFA1 in vitro at concentrations sufficient to also account for FFA1 activation in vivo. Each CLA isomer markedly increased glucose-stimulated insulin secretion in insulin-producing INS-1E cells that endogenously express FFA1 and in primary pancreatic β-cells of wild type but not FFA1(-/-) knock-out mice. Our findings establish a clear mechanistic link between CLAs and insulin production and identify the cell surface receptor FFA1 as a molecular target for CLAs, explaining their acute stimulatory effects on insulin secretion in vivo. CLAs are also revealed as insulinotropic components in widely used nutraceuticals, a finding with significant implication for development of FFA1 modulators to treat type 2 diabetes.