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microRNAs (miRNAs) are an extensive class of newly identified endogenous small regulatory molecules. Many studies show that miRNAs play a critical role in almost all biological and metabolic progresses through targeting protein-coding genes for mRNA cleavage or translation inhibition. Many miRNAs are also identified from cotton using computational and/or experimental approaches, including the next generation deep sequencing technology. However, the function of the majority of miRNAs are unclear. In this chapter, we describe a detailed method for overexpressing miR 156 in cotton using
Agrobacterium
-mediated genetic transformation. This provides an approach to investigate the function and regulatory mechanism of miRNAs in cotton.