microRNAs (miRNAs), a kind of small noncoding RNAs, are considered able to regulate expression of genes and mediate RNA silencing. miR-129-5p was shown to be a cancer-related miRNA. However, the influence of miR-129-5p in rectal adenocarcinoma (READ) development remains to be determined. Based on the TCGA data, downregulation of miR-129-5p in READ samples was observed. Manual restoration of the miR-129-5p in SW1463 and SW480 cell lines significantly inhibited invasion, migration, and proliferation of READ cell lines, while the apoptosis ability was enhanced. Meanwhile, we found E2F7 acted as a potential target of miR-129-5p and was upregulated in READ samples. E2F7 upregulation reversed the repression of miR-129-5p on READ development. Finally, in vivo experiments showed that inhibition of tumor growth in nude mice was achieved through upregulating miR-129-5p. Overall, our findings suggest increasing of miR-129-5p leads to the suppression of READ progression through regulating the expression of E2F7, which may provide novel insights into the treatment of READ. © 2020 Wiley Periodicals, Inc.