This study reports the interaction of indole-3-carbinol (I3C), which is a chemopreventive reagent, with an artificial model membrane {(dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLVs)}, using the intrinsic fluorescence properties of I3C, extrinsic fluorescence properties of Nile Red (NR), differential scanning calorimetry (DSC), dynamic light scattering (DLS), and confocal microscopy. The intrinsic fluorescence of I3C helps to provide information about its location, partitioning ability, and sensitivity toward the phase-transition temperature of liposomes, confirmed by cetylpyridinium chloride (CPC) quenching study, partition coefficient values {(4.60 ± 0.1) × 105 (solid gel phase) and (7.29 ± 0.1) × 105 M-1 (liquid crystalline phase)} and temperature-dependent emission behavior of I3C. I3C perturbs the DMPC MLVs above 15 mol %, as observed using the fluorescence properties of NR, DSC, and DLS data. This perturbation occurs as a consequence of interfacial hydration of the DMPC MLVs, which was clearly indicated by the fluorescence properties (emission intensity, fluorescence lifetime, and nonextensive distribution analysis) of NR.