Although numerous differential long noncoding RNAs (IncRNAs) have been identified, the relationship between lncRNA and colorectal cancer (CRC) still remains largely unclear. In the present study, we investigated the function and provided a possible mechanism of lncRNA FAM3D-AS1 in CRC. CCK8, transwell, and trypan blue staining were used to evaluate the proliferation, invasion, and cell death rates. Real-time PCR was used to elucidate the expression level of FAM3D-AS1. We found that lncRNA FAM3D-AS1 located in cytoplasm. Overexpression of FAM3D-AS1 significantly inhibited the cell proliferation, cell survival rates, and invaded cells. We also provided evidence that FAM3D-AS1 reversed the EMT process. Moreover, we proved that FAM3D-AS1 inhibits CRC development through NF-κB signaling pathway. Taken together, we performed functional analysis of FAM3D-AS1 and provided a possible mechanism in the development of CRC. Our study provided new targets for clinical treatment.