The maintenance of cell membrane fluidity is of critical importance for various cellular functions. At lower temperatures when membrane fluidity decreases, plants and cyanobacteria react by introducing unsaturation in the lipids, so that the membranes return to a more fluidic state. To probe how introduction of unsaturation leads to reduced membrane fluidity, a model cationic lipid dioctadecyldimethylammonium bromide (DODAB) has been chosen, and the effects of an unsaturated lipid monoolein (MO) on the structural dynamics and phase behavior of DODAB have been monitored by quasielastic neutron scattering and time-resolved fluorescence measurements. In the coagel phase, fluidity of the lipid bilayer increases significantly in the presence of MO relative to pure DODAB vesicles and becomes manifest in significantly enhanced dynamics of the constituent lipids along with faster hydration and orientational relaxation dynamics of a fluorophore. On the contrary, MO restricts both lateral and internal motions of the lipid molecules in the fluid phase (>330 K), which is consistent with relatively slow hydration and orientational relaxation dynamics of the fluorophore embedded in the mixed lipid bilayer. The present study illustrates how incorporation of an unsaturated lipid at lower temperatures (below the phase transition) assists the model lipid (DODAB) in regulating fluidity via enhancement of dynamics of the constituent lipids.