MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level. Our previous studies suggest that mir-21 functions as an oncogene and has a role in tumorigenesis, in part through regulation of the tumor suppressor gene tropomyosin 1 (TPM1). Given that TPM1 has been implicated in cell migration, in this study we further investigated the role of mir-21 in cell invasion and tumor metastasis. We found that suppression of mir-21 in metastatic breast cancer MDA-MB-231 cells significantly reduced invasion and lung metastasis. Consistent with this, ectopic expression of TPM1 remarkably reduced cell invasion. Furthermore, we identified two additional direct mir-21 targets, programmed cell death 4 (PDCD4) and maspin, both of which have been implicated in invasion and metastasis. Like TPM1, PDCD4 and maspin also reduced invasiveness of MDA-MB-231 cells. Finally, the expression of PDCD4 and maspin inversely correlated with mir-21 expression in human breast tumor specimens, indicating the potential regulation of PDCD4 and maspin by mir-21 in these tumors. Taken together, the results suggest that, as an oncogenic miRNA, mir-21 has a role not only in tumor growth but also in invasion and tumor metastasis by targeting multiple tumor/metastasis suppressor genes. Therefore, suppression of mir-21 may provide a novel approach for the treatment of advanced cancers.