Although gain of chromosome 5p is one of the most frequent DNA copy-number imbalances in cervical squamous cell carcinoma (SCC), the genes that drive its selection remain poorly understood. In a previous cross-sectional clinical study, we showed that the microRNA processor Drosha (located on chromosome 5p) demonstrates frequent copy-number gain and overexpression in cervical SCC, associated with altered microRNA profiles. Here, we have conducted gene depletion/overexpression experiments to demonstrate the functional significance of up-regulated Drosha in cervical SCC cells. Drosha depletion by RNA interference (RNAi) produced significant, specific reductions in cell motility/invasiveness in vitro, with a silent RNAi-resistant Drosha mutation providing phenotype rescue. Unsupervised hierarchical clustering following global profiling of 319 microRNAs in 18 cervical SCC cell line specimens generated two groups according to Drosha expression levels. Altering Drosha levels in individual SCC lines changed the group into which the cells clustered, with gene depletion effects being rescued by the RNAi-resistant mutation. Forty-five microRNAs showed significant differential expression between the groups, including four of 14 that were differentially expressed in association with Drosha levels in clinical samples. miR-31 up-regulation in Drosha-overexpressing samples/cell lines was the highest-ranked change (by adjusted p value) in both analyses, an observation validated by northern blotting. These functional data support the role of Drosha as an oncogene in cervical SCC, by affecting expression of cancer-associated microRNAs that have the potential to regulate numerous protein-coding genes. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.