Transfer RNAs (tRNAs) are typically considered housekeeping products with little regulatory function. However, several studies over the past 10 years have linked tRNA misregulation to cancer. We have previously reported that tRNA levels are significantly elevated in breast cancer and multiple myeloma cells. To further investigate the cellular and physiological effects of tRNA overexpression, we overexpressed tRNAi(Met) in two human breast epithelial cell lines. We then determined tRNA abundance changes and performed phenotypic characterization. Overexpression of tRNAi(Met) significantly altered the global tRNA expression profile and resulted in increased cell metabolic activity and cell proliferation. Our results extend the relevance of tRNA overexpression in human cells and underscore the complexity of cellular regulation of tRNA expression.