Mesoporous silica (MPS), a carrier for active pharmaceutical ingredients, has a wide range of particle and pore morphology. A thorough understanding of ingredients used in MPS synthesis is an important prerequisite for optimizing its physicochemical characteristics. The present study aimed to evaluate the effect of glycerol and hydrochloric acid on the characteristics of synthesized MPS. Ordered MPS materials were synthesized using the pluronic P123 template and tetraethyl orthosilicate (TEOS) precursor. A three-level factorial design was employed to study the interaction between glycerol and hydrochloric acid. The optimized MPS particles were reasonably uniform in shape (short and rod-shaped) and < 1 μm in size with a smooth surface morphology. The nitrogen adsorption–desorption analysis revealed that the uniform cylindrical pores of the prepared MPS had a diameter > 5 nm and a total surface area > 500 m2/g. With increasing acid and glycerol concentrations, the particle size of MPS decreased. However, while the glycerol increased the heterogeneity of the synthesized particles, the acid decreased it. The developed MPS was successfully loaded with a biological drug (insulin) with a 21.94% encapsulation efficiency. The MPS prepared in this study exhibits potential applications as a drug delivery carrier for drugs with a large molecular weight.