The small molecule chemical compound cinobufotalin (CB) is reported to be a potential antitumour drug that increases cisplatin (DDP) sensitivity in nasopharyngeal carcinoma. In this study, we first found that CB decreased DDP resistance, migration and invasion in lung adenocarcinoma (LUAD). Mechanistic studies showed that CB induced ENKUR expression by suppressing PI3K/AKT signalling to downregulate c-Jun, a negative transcription factor of ENKUR. Furthermore, ENKUR was shown to function as a tumour suppressor by binding to β-catenin to decrease c-Jun expression, thus suppressing MYH9 transcription. Interestingly, MYH9 is a binding protein of ENKUR. The Enkurin domain of ENKUR binds to MYH9, and the Myosin_tail of MYH9 binds to ENKUR. Downregulation of MYH9 reduced the recruitment of the deubiquitinase USP7, leading to increased c-Myc ubiquitination and degradation, decreased c-Myc nuclear translocation, and inactivation of epithelial-mesenchymal transition (EMT) signalling, thus attenuating DDP resistance. Our data demonstrated that CB is a promising antitumour drug and may be a candidate chemotherapeutic drug for LUAD patients.© 2022. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.