Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors. Of this family, PPARδ has been implicated in the pathogenesis of colorectal cancer, whereas its exact role is highly controversial as available studies yield conflicting results. Therefore, further studies using powerful and duplicable methods are needed to clarify the functions of PPARδ in colorectal cancer. RNA interference (RNAi) is such a powerful tool for specific suppression of gene expression at the posttranscriptional level. Of available RNAi methods, lentivirus has been shown to mediate RNAi most efficiently and stably based on its remarkable advantages, such as powerful transduction efficiency and a wider range of target cells, integration of expression cassette into host genomics, low immunogenicity and self-inactivated and replication-incompetent after integrating into host genomics. In this chapter, we will introduce the details of using lentivirus-mediated RNAi to specifically knockdown the expression of PPARδ in colon cancer cell lines. This method includes: designing and cloning short-hairpin RNA cassette into lentivector, constructing and titrating lentiviral particles, infecting target cells with lentivirus and assaying the knockdown efficiency.