Dicer is a protein that plays a pivotal role in the final steps of the microRNA (miRNA) processing pathway, to produce mature miRNAs from their precursor molecules. The purpose of the current study was to assess the biological and prognostic value of Dicer protein expression in breast cancer (BC). Dicer protein expression was assessed immunohistochemically in two sets of BC: (1) full-face sections of selected BC series with distinct stages of tumour progression (normal, in situ (DCIS), primary invasive BC and nodal metastases) to evaluate its differential expression. (2) Tissue microarray comprising a large and well-characterised series of unselected clinically annotated invasive BC (n = 1,174) to investigate its correlation with clinicopathological features and patient outcome. A gradual loss of Dicer protein expression was observed in malignant compared to normal breast tissues, with the loss being the least in DCIS and most prominent in metastatic malignant cells. In invasive BC, loss of Dicer expression was associated with features of aggressive behaviour including higher histological grade, loss of hormone receptor and BRCA1 protein expression and with shorter disease-free survival (DFS). Dicer expression was an independent predictor of recurrence in the aggressive HER2-positive subgroup. Moreover, loss of Dicer was predictive of better response to chemotherapy and to endocrine therapy. This study provides evidence that Dicer protein plays a role in human BC progression and behaviour, and assessment of its expression could provide prognostic information in BC including the HER2-positive class.