Cathepsin G (CG), a neutrophil serine protease, induces cell migration and multicellular aggregation of human breast cancer MCF-7 cells. It has been suggested that tumor cell aggregates are associated with tumor embolism, thus CG-induced cell aggregation may promote tumor metastasis. We have revealed that cell aggregation is caused by elevated free insulin-like growth factor (IGF)-1 in the medium, followed by activation of IGF-1 receptor (IGF-1R). However, the molecular mechanism underlying IGF-1 elevation induced by CG remains unclear. Here, we aimed to elucidate the mechanism by examining the degradative effects of CG on IGF-1, and the IGF binding proteins (IGFBPs), which interfere with the binding of IGF-1 to its receptor. CG specifically evoked MCF-7 cell aggregation at less than 1 nM in a dose-dependent manner, however, neutrophil elastase (NE), chymotrypsin, and trypsin did not. Free IGF-1 concentration was continuously elevated in the medium of cells treated with CG, whereas treatments with other serine proteases resulted in only a transient or slight increase. IGFBP-2, the predominant IGFBP in MCF-7 cells, was gradually digested by CG. CG did not cleave IGF-1 for at least 48 h, whereas other proteases completely digested it. Moreover, CG induced continuous phosphorylation of IGF-1R and Akt, whereas NE-induced phosphorylation was transient, possibly due to insulin receptor substrate (IRS)-1 digestion. These results indicated that CG-specific IGF-1 elevation in the medium is caused by digestion of IGFBP-2, not IGF-1. Hence, this study clarifies the molecular mechanism of CG-specific cell aggregation.