Abstract: The present study aimed to investigate the optimal conditions for the formation of phosphorylated fish bone collagen peptide (PFBCP)-calcium nanoparticles in a simulated gastrointestinal tract and to evaluate their calcium transport characteristics. The calcium ion concentration required for PFBCP-calcium nanoparticle formation was determined using turbidity measurements, and the nanoparticles' characteristics were examined using particle size analysis and electron microscopy. The calcium transport effect and pathway of PFBCP-calcium nanoparticles were analyzed by constructing a Caco-2 monolayer cell model. The results showed that PFBCP could form mostly spherical, uniformly dispersed peptide-calcium nanoparticles with sizes ranging from 31 to 116 nm in a gastrointestinal simulation system containing calcium chloride concentrations of 7.5~20.0 mmol/L. With an increase in calcium chloride concentration, the particle size of PFBCP-calcium nanoparticles also increased, whereas both surface charge and surface hydrophobicity decreased. Dietary components such as casein, myofibrillar protein, chondroitin sulfate, and lecithin significantly promoted the formation of PFBCP-calcium nanoparticles in the simulated gastrointestinal system (P<0.05). PFBCP-calcium nanoparticles exhibited significantly higher levels of calcium transport than did calcium chloride (P<0.05), primarily delivering calcium through the unique pathways of endocytosis and the paracellular route, which are independent of the TRPV6 calcium ion channel. Overall, PFBCP-calcium nanoparticles formed in the gastrointestinal tract demonstrated effective calcium transport capabilities, indicating promising prospects for application development.