Epidermal growth factor receptor (EGFR) is over-expressed in several human cancers. This would suggestthat inhibition of EGFR is a reasonable approach for cancer treatment. In this study we investigated EGFRblocking and its effects on the mediated signaling such as MAPK and STATb in HT29 cells. For this aim weused FITC-labeled EGFR antisense oligonucleotides encapsulated with PAMAM nanoparticles to inhibit EGFRexpression. Cellular uptake of antisense was investigated by fluorescence microscopy and flow cytometry analysis.The effect of EGFR antisense on the expression of EGFR in HT29 cells was examined by real time PCR andWestern blots, which showed that antisense encapsulated with PAMAM decreased the level of EGFR mRNAand protein. In addition, real time PCR results confirmed that EGFR inhibition had an effective role in thereduction of EGFR dependent downstream genes. In conclusion, EGFR antisense encapsulated with PAMAMnanoparticles down regulated EGFR and EGFR-mediated genes.