Peroxisome proliferator-activated receptor delta (PPAR-delta), one of three PPAR subtypes, is a lipid-sensing nuclear receptor that has been implicated in multiple processes, including inflammation and cancer. To directly establish the role of PPAR-delta in colon cancer development and progression, we selected high-affinity RNA aptamers and expressed them in several colon cancer cell lines. Nuclear-expressed aptamers efficiently inhibited PPAR-delta-dependent transcription from a synthetic peroxisome proliferator response element-driven luciferase reporter. PPAR-delta-specific aptamers suppressed transcription from natural promoters of vascular endothelial cell growth factor-A and cyclooxygenase-2. Moreover, vascular endothelial cell growth factor-A and cyclooxygenase-2 mRNA levels were significantly reduced by the PPAR-delta-specific aptamers in colon cancer cells. Most significantly, HCT116 colon cancer cells with high-level expression of PPAR-delta-specific aptamers exhibited a striking loss of tumorigenic potential. Further study on these RNA aptamers could provide an opportunity to modulate PPAR-delta-mediated colon cancer development and progression. Taken together, our results establish an important role for PPAR-delta in transcription of tumor-promoting genes, which can be specifically modulated by high-affinity RNA intramers in colon cancer cells. The RNA intramers may be further developed as specific inhibitors for cancer therapeutic strategies.