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BACKGROUND: Calcium is important in the formation of bones and teeth, cell metabolism, and other physiological activities. In this work, casein phosphopeptide‐calcium chelate (CPP‐Ca) was synthesized and the optimal process parameters for the chelation reaction were obtained. The bioavailability of calcium in CPP‐Ca was investigated by in vitro gastrointestinal simulated digestion. The existence of phytic acid and oxalic acid in the digestion system was evaluated to clarify the calcium holding ability of casein phosphopeptide (CPP). Liquid chromatography–tandem mass spectrometry (LC‐MS/MS) was used to identify oligopeptides from CPP‐Ca. RESULTS: The optimal process parameters for the chelation reaction were: peptide concentration 7.76 mgmL−1, pH 8.54, and reaction temperature 43.3 °C. The digestion in vitro results indicated that the calcium release rate of CPP‐Ca in the stomach for 2 h reached 85%, and about 50% of the ionized calcium was re‐chelated with CPP in the intestine. Phytic acid and oxalic acid could lead to a sharp decrease in soluble calcium but around 50% of the calcium was still retained in the form of chelates in the presence of CPP. The LC‐MS/MS identified 19 casein‐derived oligopeptides after digestion, and calcium modifications were found on eight peptides derived from β‐casein and αs2‐casein. CONCLUSIONS: This study clarified the excellent calcium holding capacity of CPP in the presence of phytic acid and oxalic acid. Liquid chromatography–tandem mass spectrometry also revealed peptide changes, and identified peptides that chelate with calcium. These findings provided significant insights that could be relevant to the further utilization and product development of peptide‐calcium chelate in the food industry. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]