MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate expression of target mRNA. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and considered as new therapeutic targets for cancers. In our study, we performed a gain-of-function screen using 319 miRNAs to identify those affecting cell proliferation and death in human colorectal cancer cells (DLD-1). We discovered a number of miRNAs that increased or decreased cell viability in DLD-1. They included known oncogenic miRNAs such as miR-372 and miR-373, and tumor suppressive miRNAs such as miR-124a, but also some for which this information was novel. Among them, miR-491 markedly decreased cell viability by inducing apoptosis. We demonstrated that Bcl-X(L) was a direct target of miR-491, and its silencing contributed to miR-491-induced apoptosis. Moreover, treatment of miR-491 suppressed in vivo tumor growth of DLD-1 in nude mice. Our study provides a new regulation of Bcl-X(L) by miR-491 in colorectal cancer cells, and suggests a therapeutic potential of miRNAs for treating colorectal cancer by targeting Bcl-X(L).