Most solid tumors invade peritumoral tissues as multicellular units while maintaining cell-cell interactions and epithelial characteristics, a process known as collective invasion. Our previous proteomic analysis revealed that S100-family proteins were upregulated in the PDAC secretome. Therefore, we aimed to elucidate the functional roles of these proteins in collective invasion and PDAC metastasis. To this end, we established a 3D collective invasion model using co-culture spheroids composed of PDAC cells and pancreatic stellate cells (PSCs). Using this model, we found that S100A4 contributes to collective invasion by mediating PDAC cell-PSC interactions, with S100A4 knockdown inhibiting collective invasion and S100A4 overexpression promoting it. Additionally, depletion of S100A in PDAC cells reduced trans-endothelial migration and cancer cell adhesion to endothelial cells. Mechanistically, PDAC cell-derived extracellular S100A4 promotes LAMB3 expression in PSCs. S100A4 depletion significantly reduced histological tumor aggressiveness in a pancreatic tumor orthotopic model and inhibited metastasis. To explore the therapeutic potential of these findings, we developed de novo S100A4-targeting inhibitory peptides using AI-guided structure modeling and confirmed their inhibitory effects on collective invasion. Collectively, our findings identify S100A4 as a critical driver of collective invasion and PDAC metastasis and support extracellular S100A4 as a potential target for AI-guided therapeutic intervention.