Simvastatin belongs to the statin family of cholesterol lowering drugs which act as competitive inhibitors of HMG-CoA reductase, the rate-determining enzyme in cholesterol biosynthesis pathway. Simvastatin is a semi-synthetic, highly lipophilic statin, and has several side effects. Since HMG-CoA reductase is localized in the endoplasmic reticulum, orally administered simvastatin needs to cross the cellular plasma membrane to be able to act on HMG-CoA reductase. With an overall goal of exploring the interaction of simvastatin with membranes, we examined the effect of simvastatin on the organization and dynamics in membranes of varying phase, in a depth-dependent manner. For this, we employed DPH and TMA-DPH, which represent fluorescent membrane probes localized at two different locations (depths) in the membrane. Analysis of fluorescence anisotropy and lifetime data of these depth-specific probes in membranes of varying phase (gel/fluid/liquid-ordered) showed that the maximum membrane disordering was observed in gel phase, while moderate effects were observed in liquid-ordered phase, with no significant change in membrane order in fluid phase membranes. We conclude that simvastatin induces change in membrane order in a depth-dependent and phase-specific manner. These results provide novel insight in the membrane interaction of simvastatin and could be crucial for understanding its pharmacological effect.