Cachexia, characterized by loss of skeletal muscle mass and function, is estimated to inflict the majority of patients with oesophageal squamous cell carcinoma (ESCC) and associated with their poor prognosis. However, its underlying mechanisms remain elusive. Here, we developed an ESCC-induced cachexia mouse model using human xenograft ESCC cell lines and found that ESCC-derived extracellular vesicles (EVs) containing prolyl 4-hydroxylase subunit beta (P4HB) induced apoptosis of skeletal muscle cells. We further identified that P4HB promoted apoptotic response through activating ubiquitin-dependent proteolytic pathway and regulated the stability of phosphoglycerate dehydrogenase (PHGDH) and subsequent antiapoptotic protein Bcl-2. Additionally, we proved that the P4HB inhibitor, CCF642, not only rescued apoptosis of muscle cells in vitro, but also prevented body weight loss and muscle wasting in ESCC-induced cachexia mouse model. Overall, these findings demonstrate a novel pathway for ESCC-induced muscle wasting and advocate for the development of P4HB as a potential intervention target for cachexia in patients with ESCC. © 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.