Expression and secretion of procathepsin D (pCD) increases proliferation, metastasis and progression of breast cancer but the structural moiety by which pCD exerts these effects is still ambiguous. Here, we present data on a series of pCD stable mutants to identify the pCD region that mediates this mitogenic effect. Mutations affecting the region of the activation peptide (AP) were studied together with catalytic and glycosylation mutants. Mitogenic effect was evaluated using in vitro invasion and proliferation assays and in vivo by determining the tumorigenic potential. The catalytic mutants and glycosylation mutants of pCD continued to display enhanced cell proliferation, invasion and tumorigenicity similar to stable transfectants of native pCD, suggesting that neither the proteolytic activity nor the sugar moieties contribute to the mitogenic effect. However, stable transfectants of pCD lacking its AP and with various mutations in the 27-44 amino acid region of AP, failed to show enhanced cell proliferation or invasion in vitro and tumor growth in vivo, establishing the importance of AP region. Our study concludes that the entire 27-44 amino acid region of AP is necessary for the stimulatory actions of pCD on breast cancer cells.