The leading cause of death in cancer patients is cancer metastasis, for which there is no effective treatment. MicroRNAs (miRNA) have been shown to play a significant role in cancer metastasis through regulation of gene expression. The adenovirus type 5 E1A (E1A) is associated with multiple tumor-suppressing activities including the inhibition of metastasis, and E1A gene therapies have been tested in several clinical trials. However, the mechanisms involved in E1A-mediated tumor-suppressing activities are not yet completely defined. Here, we showed that E1A downregulated the expression of the miRNA miR-520h, which was critical for E1A-mediated cancer cell mobility and in vitro invasion activity. In addition, we identified a signal cascade, namely, E1A-->miRNA-520h-->PP2A/C-->IkappaB kinase-->NF-kappaB-->Twist, in which E1A inhibited the expression of Twist through downregulation of miR-520h and the signal cascade. Our results indicated a functional link between miR-520h and tumorigenicity/invasive ability and provided a new insight into the role of E1A-mediated miRNA regulation in tumor suppression. Therefore, the results identified a new cascade of E1A-mediated tumor suppression activity via downregulation of miRNA-520h expression.