MicroRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene silencing by binding to complementary target mRNAs and recruiting the miRNA-containing ribonucleoprotein complexes to the mRNAs. However, the molecular basis of this silencing is unclear. Here, we show that human Ago2 associates with the cap-binding protein complex and this association is mediated by human eIF4GI, a scaffold protein required for the translation initiation. Using a cap photo-crosslinking method, we show that Ago2 closely associates with the cap structure. Taken together, these data suggest that eIF4GI participates in the miRNA-mediated post-transcriptional gene silencing by promoting the association of Ago2 with the cap-binding complex.