Recent in vivo studies suggest a role for relaxin-3 in feeding and stress. To further elucidate the function of relaxin-3 in the central nervous system, we have employed a complementary approach, based on RNA interference, to modulate relaxin-3 expression. We have designed, constructed, and characterized three microRNAs (miRNAs) targeting different regions of the relaxin-3 transcript. These were tested to determine the amount of miRNA required to achieve the greatest knockdown and for their ability to reduce the expression of relaxin-3 in transfected HEK293Tcells. All miRNA constructs significantly reduced relaxin-3-induced cAMP responses; however, miR499 was most effective. This should be a useful tool for in vitro studies and central targeting of relaxin-3 in vivo using viral delivery systems.