Pepsin is the first protease that food proteins encounter in the digestive tract. However, most of the previous studies on the enzymatic kinetics of pepsin were based on the hydrolysis of small synthetic peptides, due to the limitations in methodology and the complexity of protein substrate. To better understand the role of pepsin in protein digestion, we used isothermal titration calorimetry to study the enzymatic kinetics of pepsin with bovine serum albumin as the substrate. We found that pepsin has a higher catalytic rate at lower pH, while its affinity to substrate is lower. At the same pH, pepsin has lower activity and affinity at higher ionic strengths. We found contrasting kinetic parameters for pepsin-catalyzed hydrolysis of bovine serum albumin and of small synthetic peptides. Time-dependent kinetics also showed that pepsin has lower efficiency towards intermediate peptides during hydrolysis.