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Background: Ewing sarcoma-associated transcript 1 (EWSAT1) has been reported to be a pivotal modulator in a series of cancers. However, the function of EWSAT1 in colorectal cancer (CRC) has not been elaborated. This study aimed to explore the role of EWSAT1 in CRC progression and the underlying mechanisms. Methods: The expression patterns of EWSAT1, miR-326 and FBXL20 were examined by qCRCR. Si-EWSAT1 was transfected to study the effects of EWSAT1 on cell proliferation and metastasis. Rescue experiments were performed to investigate the underlying mechanisms in vitro. Xenograft models were used to evaluate the role of EWSAT1 in vivo. Results: We found that EWSAT1 was highly expressed in CRC tissues and cell lines and associated with poor overall survival. In vitro, knockdown of EWSAT1 suppressed the cell proliferation, migration and invasion. Moreover, miR-326 was found to be a target of EWSAT1, and miR-326 inhibitor could partially reverse the effects on CRC cell progression induced by si-EWSAT1. Subsequently, we validated FBXL20 as a vital downstream target for miR-326, and EWSAT1 positively regulated FBXL20 via miR-326 in vitro. In addition, these findings were confirmed by in vivo experiments. Conclusion: Taken together, the data showed that EWSAT1 promoted CRC progression via targeting miR-326/FBXL20 pathway, which might provide a novel therapeutic target for CRC treatment. [ABSTRACT FROM AUTHOR]