Small interfering RNA (siRNA) have been proved to be an effective tool for treating diseases caused by gene expression abnormalities. However, clinical relevance of siRNA technology is compromised by multiple obstacles, such as lack of cellular specificity, low efficiency of cellular uptake and/or poor cytoplasmic release. We have previously demonstrated that natural capability of certain immune cells to recognize non-methylated CpG motifs in DNA oligonucleotides can be harnessed for cell-specific siRNA delivery. The siRNA molecules conjugated to CpG oligonucleotides (CpG-siRNA) are efficiently internalized and bind to the intracellular Toll-like receptor 9 (TLR9) in human and mouse dendritic cells as well as in certain TLR9-positive tumor cells. We designed a CpG-siRNA targeting Stat3 (signal transducer and activator of transcription-3), a transcription factor, which is constitutively activated in majority of mouse and human cancers. Oncogenic Stat3 signaling regulates cancer cell proliferation, survival and promotes tumor immunoresistance. We have previously shown that CpG-Stat3 siRNA can eliminate Stat3 signaling in tumor-infiltrating myeloid cells, resulting in potent antitumor immunity. In the current study, we investigated the intracellular mechanism of action of CpG-siRNA conjugates. We have demonstrated that internalized CpG-siRNAs are recruited into endosomes by dynamin-dependent mechanism and bind to endosomal TLR9 receptor. Further events include uncoupling of siRNA from the CpG moiety by Dicer endonuclease, and rapid translocation of siRNA to endoplasmic reticulum for recruitment into functional RISC (RNA interference silencing complex). We confirmed the target gene silencing both in vitro and in vivo, and proved that it occurs through RNA interference mechanism. Our current results indicate that downstream effectors of TLR9 pathway may be involved in RISC assembly and/or activation. Overall, the CpG-siRNA strategy provides a novel opportunity for overcoming cancer immunoresistance with translational potential for therapy of various malignancies.