AbstractElevated levels of RNA binding protein HuR were found in various human cancers. However, the mechanisms underlying HuR over‐expression in cancers have not been fully elucidated. Here, we show that miR‐16 acts as a novel post‐transcriptional regulator for HuR. Knockdown of miR‐16 increased HuR protein levels in MDA‐MB‐231 cells, while over‐expression of pre‐miR16 reduced HuR expression. Neither knockdown nor over‐expression of miR‐16 could alter the mRNA levels of HuR. Instead, knockdown of miR‐16 increased the level of de novo synthesized HuR protein. Importantly, mechanistic studies showed that miR‐16 associated with the 3′UTR of HuR, and knockdown of miR‐16 markedly increased the luciferase activity of a HuR 3′UTR‐containing reporter. We further demonstrate that the level of miR‐16 was inversely correlated with HuR protein level in human breast carcinoma. Together, our results suggest an important role of miR‐16 in regulating HuR translation and link this regulatory pathway to human breast cancer. J. Cell. Biochem. 111: 727–734, 2010. © 2010 Wiley‐Liss, Inc.