The effects of calcium ion on the structural properties of casein micelles in the course of heat treatment were synthetically examined by non-structure-invasive spectrometry. The hydrophobicity, reflected by extrinsic fluorescence (ANS fluorescence), was positively correlated with the concentration of the calcium ion, within the range of 0 to 12 mmol·L-1. Meanwhile, the turbidity and stability of casein micelles also increased with the growth of calcium concentrations. However, opposite results were observed for hydrodynamic diameter and polydispersity index. Compared with the calcium ion, the calcium-chelator (citrate) has an opposite effect on the structural characteristics of casein micelles. Within the calcium concentrations range of 0 to 12 mmol·L-1, the hydrophobicity, stability and turbidity were negatively correlated with the concentration of the calcium ion, nevertheless, opposite results were observed for hydrodynamic diameter and polydispersity index. All the results indicate that the calcium ion could be used to modify the structures of casein micelles during heat heatment.