Accumulating evidence has revealed that long noncoding RNAs (lncRNAs) play essential roles in regulating cellular process of various cancers. There have been many studies on the biological functions of lncRNAs in colorectal cancer (CRC). In this research, we explored the role and mechanism of lncRNA ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) in CRC. Here, we detected OTUD6B-AS1 expression in CRC tissues and cells by RT-qPCR. Functional experiments were performed to test alterations in different cellular processes. Moreover, to verify the binding ability among the indicated RNA molecules, we carried out RIP, RNA pull-down and luciferase reporter assays. According to our data, OTUD6B-AS1 expression was low in CRC tissues and cells. Functionally, overexpression of OTUD6B-AS1 inhibited cell proliferation, migration, invasion and EMT, and promoted cell apoptosis. Bioinformatic analysis and mechanistical experiments confirmed that OTUD6B-AS1 could act as a competitive endogenous RNA (ceRNA) to upregulate Proline-Rich Nuclear Receptor Coactivator 2 (PNRC2) expression by sequestering miR-21-5p. Further rescue experiments validated the inhibitory function of the OTUD6B-AS1/miR-21-5p/PNRC2 axis in cellular process of CRC. Overall, OTUD6B-AS1 inhibits cellular development in CRC by sponging miR-21-5p and upregulating PNRC2, providing a novel insight into the exploration on CRC treatment.