Background <p>The mammalian STE20-like kinase 4 (MST4) has been implicated as a potential oncogene in various cancers, including pancreatic cancer (PC). However, the precise mechanisms through which MST4 promotes PC malignancy remain incompletely understood.</p> Methods <p>A Biotin identification (BioID)-based proximity labeling approach was employed to identify key MST4-interacting molecules in human PANC-1 pancreatic cancer (PC) cells, followed by cell migration assay to validate the synergistic promotional effect of the MST4-interacting complex on PC cell migration. Direct binding between MST4 and its interacting partners was confirmed via microscale thermophoresis (MST) and isothermal titration calorimetry (ITC), with further validation through X-ray crystallographic structural analysis. The efficacy of rationally designed peptides targeting this interaction was evaluated in both in vitro cell models and in vivo xenograft mouse models.</p> Results <p>In this study, we demonstrate that MST4 forms a phosphorylation-dependent complex with 14-3-3ζ, leading to Yes-associated protein (YAP) activation and synergistic enhancement of PC cell migration. We observed significant upregulation of both MST4 and 14-3-3ζ in PC patient samples, which correlated strongly with YAP activation and poor prognosis. Structural analysis revealed the detailed interface of the MST4–14-3-3ζ complex, facilitating the rational design of peptide inhibitors that disrupt this interaction. These peptides effectively suppressed YAP activation and exhibited potent anti-tumor effects both in vitro and in vivo.</p> Conclusions <p>Our findings establish the MST4–14-3-3ζ complex as a critical regulator of YAP signaling that synergistically promotes PC cell migration. Furthermore, rationally designed peptides targeting this MST4–14-3-3ζ interaction represent a promising therapeutic strategy for pancreatic cancer and warrant further clinical exploration.</p> Graphical Abstract <p></p>