MicroRNAs, key posttranscriptional regulators of eukaryotic gene expression, play important roles in plant development and response to stress. In this study, a soybean gma-MIR394a gene was functionally characterized, especially with regard to its role in drought stress resistance. Expression analysis revealed that gma-MIR394a was expressed differentially in various soybean tissues and was induced by drought, high salinity, low temperature stress, and abscisic acid treatment in leaves. One target gene of gma-miR394a, Glyma08g11030, was predicted and verified using a modified 5' RLM-RACE (RNA ligase-mediated rapid amplification of 5' cDNA ends) assay. Overexpression of gma-MIR394a resulted in plants with lowered leaf water loss and enhanced drought tolerance. Furthermore, overexpression of gma-MIR394a in Arabidopsis reduced the transcript of an F-box gene (At1g27340) containing a miR394 complementary target site. These results suggest that the gma-MIR394a gene functions in positive modulation of drought stress tolerance and has potential applications in molecular breeding to enhance drought tolerance in crops. (C) 2012 Published by Elsevier Inc.