Antisense oligonucleotides present a powerful means to inhibit expression of specific target genes. They are short, single-stranded oligodeoxynucleotides (ODNs) that bind to complementary mRNA via Watson-Crick base pairing. However, poor cellular delivery impedes their widespread utilization in therapy and biotechnological research. Numerous delivery agents have already been developed to enhance their cellular uptake while also protecting them from degradation. Polyethyleneimine (PEI) is one such cationic polymer that has been widely studied for its utilization in plasmid DNA delivery for gene transfer. However, there are relatively fewer systematic investigations of the use of PEI for delivering ODNs of various chemistries. In our previous work, when several PEI molecular weights (MWs) were employed to deliver PS ODNs, we found that while lower PEI MWs were ineffective in delivering phosphorothioate (PS) ODNs, complexes of higher MW PEI and PS ODNs were very efficient in eliciting an antisense response. However, the toxicity of higher PEI MWs offsets their use as effective ODN carriers. Here, we show that it is possible to tune the effectiveness of the vector by modifying the DNA backbone chemistry. By reducing the degree of phosphorothioate substitution, we are able to dramatically improve the efficiency of lower PEI MWs as ODN carriers. For this study, we chose a series of five ODN chemistries that vary in the degree of PS end substitution from 0 to 100%. We studied the ability of these ODNs to form complexes with a panel of molecular weights of branched PEI. Further, we evaluated their effectiveness in delivering ODNs to cells. We accomplish this by measuring simultaneously the dynamics of both ODN uptake and antisense inhibition in a stably pd1EGFP expressing cell line using a cellular assay based on single cell fluorescence measurements. To provide a mechanistic understanding for our results, we draw explanations from various assays that mimic key intracellular processing steps. We measure the rates of cellular internalization of complexes, the antisense response on perturbation of the endocytic release rates with endocytic pH buffering reagents such as chloroquine and ammonium chloride, and also determine the ability of the complexes to release DNA by measuring the in vitro rates of dissociation of complexes in the presence of a competing anionic entity. The results from our work highlight several key factors that affect PEI mediated delivery of AS ODNs. In particular, we show that the same polymer can vary drastically in its ability as a delivery carrier depending on the chemistry of the cargo DNA. Further, we find that the extent and time scale of observed antisense effects is distinctly dependent on the particular combinations of the ODN chemistry and polymer MWs and that free polymer present when complexes are formulated at higher charge ratios can play a role in intracellular delivery. By correlating the PEI MW & ODN chemistry with the observed antisense effects, we are able to draw insightful structure-property relationships that will aid the rational design of ODN carriers.