Although tumour budding is an adverse prognostic factor for many cancer types, the molecular mechanisms governing this phenomenon are incompletely understood. Therefore, understanding the molecular basis of tumour budding may provide new therapeutic and diagnostic options. We employ digital image analysis to demonstrate that the number of tumour buds in cytokeratin-stained sections correlates with patients having lymph node metastases at diagnosis. The tumour bud count was also a predictor of overall survival, independent of TNM stage. Tumour buds and paired central tumour areas were subsequently collected from oral squamous cell carcinoma (OSCC) specimens using laser capture microdissection and examined with RNA sequencing and miRNA-qPCR arrays. Compared with cells from the central parts of the tumours, budding cells exhibited a particular gene expression signature comprising factors involved in epithelial-to-mesenchymal transition (EMT) and activated TGF-β signaling. Transcription factors ZEB1 and PRRX1 were upregulated concomitantly with the decreased expression of mesenchymal-to-epithelial (MET) transcription factors (e.g., OVOL1) in addition to Krüppel-like factors and Grainyhead-like factors. Moreover, miR-200 family members were downregulated in budding tumour cells. We used immunohistochemistry to validate five markers of the EMT/MET process in 199 OSCC tumours, as well as in situ hybridization in 20 OSCC samples. Given the strong relationship between tumour budding and the development of lymph node metastases and an adverse prognosis, therapeutics based on inhibiting the activation of TGF-β signaling may prove useful in the treatment of OSCC