Polyunsaturated fatty acids have pronounced effects on membrane proteins, as they modulate important physical properties of the lipid bilayer such as the lateral pressure profile. To understand the biophysical basis of this influence, we studied how polyunsaturated lipids modulate the gating behaviour of the mechanosensitive channels MscL and MscS. We found two distinct effects on the gating of these channels. Whilst MscL responds primarily to membrane thinning induced by polyunsaturated lipids, MscS is modulated by the change of lateral pressure caused by the unsaturation. To characterize the change of mechanical properties of these lipids, which are responsible for the observed modulation of the channel gating, we employed solid state 13C NMR on oriented membranes. Segmental order parameters for up to threefold polyunsaturated lipids were obtained from 13C-1H dipolar couplings measured using separated local field experiments. Using a dedicated geometrical model, we were able to determine the membrane thickness and lateral pressure profile of bilayers of polyunsaturated lipids, and correlate these membrane properties with the observed effect on mechanosensitive channels.