Phytic acid (PA) is an antinutrient that can affect calcium (Ca2+) and nutrient absorption in the human body. In this study, a novel egg yolk protein hydrolysate (EYPH) with high Ca2+-binding ability was prepared. The interaction between EYPH, PA and Ca2+ and the influence of these interactions on protein digestibility and Ca2+ transport was studied. Isothermal titration calorimetry (ITC) technology was used to explore the interaction mechanism between EYPHs, PA and Ca2+ at pH 2.0 and 7.0. Results showed that the binary interactions (PA and Ca2+, Ca2+ and EYPHs, EYPHs and PA) and ternary interaction occurred (PA, Ca2+ and EYPHs) at pH 7.0. However, under the condition of pH 2.0, only the binary interaction between EYPH and PA was performed and the ternary complex was not found. The above interaction significantly (p < 0.05) influenced protein digestibility and Ca2+ transport in vitro. PA significantly (p < 0.05) decreased the digestibility of EYPH and an increase was seen after the calcium chloride addition in the PA-EYPH reaction system. Besides, PA significantly (p < 0.05) decreased the Ca2+ transport (77%) in the small intestine, but it presented a significant (p < 0.05) increase after the EYPH addition (3.62 time). These results can provide important insights for studying the interactions, protein behavior and Ca2+ uptake of complex mixtures.