Sequencing of whole genomes has provided new perspectives into the blueprints of diverse organisms. Knowing the sequences, however, does not always tell us much about the function of the genes that regulate development and homeostasis. RNA interference (RNAi) is becoming the method of choice for gene function analysis in cells and whole organisms. Here we review the approaches available to perform RNAi experiments in mammalian cells and in mice. We discuss usage of RNAi in cancer research and as a possible therapeutic tool for cancer treatment.