Insulin entrapment within a monoolein-based reverse hexagonal (H-II) mesophase was investigated under temperature-dependent conditions at acidic (pH 3) and basic (pH 8) conditions. Studying the structure of the host H-II system and the interactions of insulin under temperature-dependent conditions has great impact on the enhancement of its thermal stabilization and controlled release for the purposes of transdermal delivery. Small angle X-ray spectroscopy (SAXS) measurements show that pH variation and/or insulin entrapment preserve the hexagonal structure and do not influence the lattice parameter. Attenuated total reflection Fourier transform spectroscopy (ATR-FTIR) spectra indicate that, although insulin interacts with hydroxyl groups of GMO in the interface region, it is not affected by pH variations. Hence different microenvironments within the H-II mesophase were monitored by a computer-aided electron paramagnetic resonance (EPR) analysis using 5-doxylstearic acid (5-DSA) as a pH-dependent probe. The microviscosity, micropolarity, order of systems, and distribution of the probes in different microenvironments were influenced by three factors: temperature, pH, and insulin solubilization. When the temperature is increased, microviscosity and order parameters decreased at both pH 3 and 8, presenting different decrease trends. It was found that, at pH 3, the protein perturbs the lipid structure while "pushing aside" the un-ionized 5-DSA probe to fit into the narrow water cylinders. At the interface region (pH 8), the probe was distributed in two differently structured environments that significantly modifies by increasing temperature. Insulin loading within the H-II mesophase decreased the order and microviscosity of both the microenvironments and increased their micropolarity. Finally, the EPR analysis also provides information about the unfolding/denaturation of insulin within the channel at high temperatures.