SMAD proteins are the core players of the transforming growth factor-beta (TGFβ) signaling pathway, a pathway which is involved in cell proliferation, differentiation and migration. On the other hand, hsa-miRNA-590-5p (miR-590-5p) is known to have a negative regulatory effect on TGFβ signaling pathway receptors. Since, RNAhybrid analy- sis suggested SMAD3 as a bona fide target gene for miR-590, we intended to investigate the effect of miR-590-5p on SMAD3 transcription.In this experimental study, miR-590-5p was overexpressed in different cell lines and its increased expression was detected through quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Western blot analysis was then used to investigate the effect of miR-590-5p overexpression on SMAD3 protein level. Next, the direct interaction of miR-590-5p with the 3´-UTR sequence of SMAD3 transcript was investigated using the dual luciferase assay. Finally, flow cytometery was used to inves- tigate the effect of miR-590-5p overexpression on cell cycle progression in HeLa and SW480 cell lines.miR-590-5p was overexpressed in the SW480 cell line and its overexpression resulted in significant reduction of the SMAD3 protein level. Consistently, direct interaction of miR-590-5p with 3´-UTR sequence of SMAD3 was detected. Finally, miR-590-5p over- expression did not show a significant effect on cell cycle progression of Hela and SW480 cell lines.Consistent with previous reports about the negative regulatory effect of miR-590 on TGFβ receptors, our data suggest that miR-590-5p also attenuates the TGFβ signaling pathway through down-regulation of SMAD3.