Abstract Background: microRNAs (miRNAs) are 19 to 24 nucleotide sequences that regulate cell proliferation, differentiation, and apoptosis. miRNA-181b is involved in tumor initiation and progression of several human malignancies including colorectal cancers (CRCs). Thus, we sought to determine the prognostic value of miRNA-181b in CRCs. Methods: Expression of miRNA-181b was quantified, by quantitative PCR (q-RT-PCR), in normal (benign colonic epithelia) tissues and in their corresponding CRC archival tissue samples collected from 345 patients. The expression levels were correlated with patient overall survival. Putative molecular targets of miRNA-181b were identified by in silico analysis. miRNA-181b mediated, post-transcriptional regulation of these targets was assessed in CRC cell lines (SW480, HCT116 p53-/- and HCT116 +/+) by transfection studies. The targets were validated by measuring their mRNA expression levels, by use of qRT-PCR, in CRC tissues that exhibited elevated levels of miRNA-181b as well as by cloning techniques to assess a direct interaction between miRNA-181b and 3’UTRs of its target genes using luciferase assays. Results: Survival analysis indicated that increased expression of miRNA-181b was associated with poor prognosis for CRCs (log rank, p=0.004). Fourteen putative molecular targets of miRNA-181b were identified by in silico analysis, and miRNA-181b mediated post-transcriptional regulation of these targets was validated in CRC cell lines by q-RT-PCR. Integrin alpha-3 was up-regulated, and BCL2LII and TNFα were down-regulated upon overexpression of miRNA-181b in vitro. Also, these targets were validated in CRC tissues that exhibited elevated miRNA-181b levels. The direct interaction of miRNA-181b with the target gene 3’UTRs was validated with luciferase assays. Conclusions: These findings demonstrate that miRNA-181b regulates expression of Integrin alpha-3, BCL2LII, and TNFα; and is a prognostic marker for colorectal cancers. Citation Format: Liselle Bovell, Balananda-Dhurjati K. Putcha, Divya Devadasan, Sejong Bae, William E. Grizzle, Upender Manne. Evaluation of the prognostic value of miRNA-181b and its target identification and validation in colorectal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2013-1944