Protein is essential to human health, but its digestion kinetics in varied structures are not yet well understood. We previously found different kinetics of protein hydrolysis in solution and in gels, and we hypothesized that the difference stemmed from the steric hindrance of gel structure to the diffusion of pepsin and its hydrolysates. To better understand the pepsin diffusivity in food matrices and its effect on digestion, we determined the diffusivity of pepsin in water and in whey protein isolate (WPI) gels by fluorescence correlation spectroscopy (FCS). We estimated the pepsin concentration gradient during digestion based on the determined diffusivity, which showed that the pepsin is constrained within a thin layer from the gel surface. Gel composition analysis confirmed this constraint: peptides as protein fragments were observed only in the first 2 mm of the WPI gels after 6 h of in vitro gastric digestion. Scanning electron microscopy indicated that pepsin loosened the microstructure of whey protein gel surfaces, which may accelerate pepsin diffusion and consequently gel surface disintegration. We conclude that the mode of whey protein gel digestion is determined by the summed effect of diffusion limitation, hydrolysis rate and microstructure transformation.