MicroRNAs (miRNAs) are novel small noncoding RNA molecules that regulate gene expression at the post transcriptional level. Compelling evidence shows that there are causative links between miRNAs deregulation and cancer development and progression. This study aims to explore the functions of miR-16-1 on proliferation, apoptosis, motility, and invasion of glioma cells. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression of miR-16-1 in normal brain tissues and two glioma cell lines, including U251 and U87. CCK-8, Annexin V/FITC (fluorescein isothiocyanate), wound healing, and transwell assays were used to evaluate the functions of miR-16-1 that involves cell proliferation, apoptosis, motility, and invasion. In addition, we conducted qRT-PCR to examine mRNA expression levels of Zyxin, one of putative target genes of miR-16-1, in U251 glioma cells after transfecting with miR-16-1 mimics. As a result, miR-16-1 expression level was lower in U251 and U87 cells than normal brain tissues. After miR-16-1 was upregulated in U251 cells, cellular proliferation was notably attenuated but cell apoptosis was not significantly increased. Moreover, overexpression of miR-16-1 attenuated migration and invasion of glioma cells, and U251 cells transfected with miR-16-1 showed significantly lower endogenous mRNA levels of Zyxin than those transfected with nonspecific control miRNA or mock (P < 0.05). In summary, we demonstrated that miR-16-1 expression was markedly decreased in human glioma cell lines, and for the first time, described the roles of miR-16-1 in cellular proliferation, migration, and invasion abilities of high-invasive glioma cells, and suggested that Zyxin may be one of putative target genes of miR-16-1. Anat Rec, 296:427–432, 2012. © 2012 Wiley Periodicals, Inc.