Aim Polyunsaturated fatty acids have been reported to reduce neuronal excitability, in part by promoting inactivation of voltage‐gated sodium and calcium channels. Effects on neuronal potassium channels are less explored and experimental data ambiguous. The aim of this study was to investigate anti‐excitable effects of polyunsaturated fatty acids on the neuronal M‐channel, important for setting the resting membrane potential in hippocampal and dorsal root ganglion neurones. Methods Effects of fatty acids and fatty acid analogues on mouse dorsal root ganglion neurones and on the human KV7.2/3 channel expressed in Xenopus laevis oocytes were studied using electrophysiology. Results Extracellular application of physiologically relevant concentrations of the polyunsaturated fatty acid docosahexaenoic acid hyperpolarized the resting membrane potential (−2.4 mV by 30 μm) and increased the threshold current to evoke action potentials in dorsal root ganglion neurones. The polyunsaturated fatty acids docosahexaenoic acid, α‐linolenic acid and eicosapentaenoic acid facilitated opening of the human M‐channel, comprised of the heteromeric human KV7.2/3 channel expressed in Xenopus oocytes, by shifting the conductance‐vs.‐voltage curve towards more negative voltages (by −7.4 to −11.3 mV by 70 μm). Uncharged docosahexaenoic acid methyl ester and monounsaturated oleic acid did not facilitate opening of the human KV7.2/3 channel. Conclusions These findings suggest that circulating polyunsaturated fatty acids, with a minimum requirement of multiple double bonds and a charged carboxyl group, dampen excitability by opening neuronal M‐channels. Collectively, our data bring light to the molecular targets of polyunsaturated fatty acids and thus a possible mechanism by which polyunsaturated fatty acids reduce neuronal excitability.