We studied the effects of κ-casein (κ-CN) dissociation from casein micelles upon heating on cheese curd formation. Cheese curd was formed by the addition of chymosin to unheated and heated (80℃, 30 min) defatted milk. In heated defatted milk, cheese curd was not formed; however, the amount of free glycomacropeptides (GMPs) and the degree of formation of para-κ-CN were the same in both the unheated and heated defatted milk. In addition, because calcium ions insolubilize upon heating, the calcium ion concentration in the heated defatted milk was adjusted to its equivalent amount in the unheated defatted milk; however, cheese curd was not formed in this case either. Therefore, chymosin was added to the casein micelles, which dissociated κ-CN. As a result, the amount of free GMP was significantly less (p < 0.05) than that in the native casein micelles. The results suggest that the quantity of formation of para-κ-CN on the micelle surface decreases due to κ-CN dissociation from the casein micelles.