Among the two-pore domain K+ channels (K2P), TREK-1 channel is one of the most studied K2P channel members due to its involvement in cardio- and neuroprotection but it suffers from a poor specific pharmacological profile. TREK-1 channel is polymodulated by voltage, pH changes, membrane stretch and polyunsaturated fatty acids (PUFAs) and generates an outwardly rectifying current. The PUFAs-mediated activation is supposedly correlated to the length of the carbon chain suggesting a link between FAs membrane insertion and a stretch effect through an increase of membrane tension. The aim of this study is to test the effects of different fatty acids, especially polyunsaturated fatty acids, to delineate a possible mechanism of action to explain the activation of ITREK-1. In this study, using the patch-clamp technique in ruptured whole-cell configuration, we compared the effects of FAs, from stearic acid (C18:0, 18 carbons; 0 double bound) to docosahexaenoic acid (C22:6, 22 carbons; 6 double bounds) on ITREK-1 in a HEK293 cell line overexpressing the human TREK-1 channel. C18:0 and C18:3 failed to activate ITREK-1. Surprisingly, it was the shortest PUFA, linoleic acid (LA C18:2) that produced the largest activated ITREK-1. At 0 mV, ITREK-1 was increased 32.4 ± 5.4 folds by LA (C18:2); DHA(C22:6): 29.7 ± 4.1 and AA(C20:4): 14.7 ± 1.9. Since the activation kinetic was fast (in the range of 1.5 min) and the reversibility immediate when FA solution was washed, we supposed that FAs interact directly with TREK-1 rather than FAs membrane insertion changing membrane tension. Some FAs linearized the current-voltage relationship with a calculated linearization index (LI) greater than 0.9, for C18:2, C22:5w6, C22:6, while the rectifier current was maintained for C20:5, C20:4, C20:5, C22:5w3 FAs (LI < 0.75). We propose that the observed linearization results from an activation process involving a K + -flux gating mechanism. Taken together, all these data suggest that the activation of TREK-1 by PUFAs is not a consequence of change in membrane tension but rather more complex than previously assumed. A better understanding of the TREK-1 channels activation mechanisms will help to develop more efficient activators in the perspective of a use to protect the heart following an infarction.