Plasmin-induced hydrolysis of casein in milk can lead to many defects including proteolysis, age gelation, and bitterness. We hypothesized that the susceptibility of casein micelles to plasmin can be affected by its micellar structure and modification of the lysine residues on caseins. Different levels of dissociation of the casein micelle structure were achieved by calcium chelation using EDTA and by succinylation by attaching a succinate group at the ε-amino group of lysine residues, leading to the formation of succinyl-lysine. The target sites for succinylation were identified using liquid chromatography-tandem mass spectrometry. Changes in the particle size and levels of casein micelle dissociation in skim milk were determined using a Zetasizer and sodium dodecyl sulfate PAGE, respectively. The subsequent plasmin hydrolysis was monitored by quantifying the hydrolyzed product using reverse phase high performance liquid chromatography. The EDTA-induced calcium chelation and dissociation of the casein micelle increased plasmin-induced hydrolysis. In contrast, succinylation had an inhibitory effect on plasmin-induced hydrolysis. Calcium chelation and succinylation of the skim milk resulted in dissociation of caseins from the casein micelle, a decrease in turbidity, extensive unfolding, and expansion of the polypeptide chain; all of these changes collectively reduced steric hindrance and made the protein more readily hydrolyzed by plasmin. However, the formation of succinyl−lysine rendered β-casein unrecognizable to the substrate-binding pocket of plasmin, resulting in a nonlinear decrease in the hydrolysis because of the competitive effect of micelle dissociation. These results clearly indicate the importance of the casein micelle structure and its susceptibility to plasmin action. They will be useful in understanding and controlling the plasmin-induced hydrolysis of milk proteins in food systems.