Background: In our previous study, HSA-PEI demonstrated high pDNA transfection efficiency and low cytotoxicity. Materials and Methods: In this study, the relationship between albumin receptors and the high pDNA transfection efficiency of HSA-PEI was investigated by fluorescent microscopy and flow-cytometer in A549 cells. Results: According to our results, the presence of an albumin receptor on A549 cells was confirmed via the application of a fluorescent tracer, FITC-HSA, and the dose-dependent competition of HSA. The transfection efficiency of HSA-PEI/pEGFP showed a dose-dependent inhibition when different amounts of HSA were added to the culture medium of the A549 cells. However, the inhibitory effect of HSA did not affect the transfection efficiency of some cationic transfection enhancement reagents, such as lipofectamine, jetPEI-RGD or PEI; their transfection was a result of contact between the positively charged reagents and the negatively charged cell surface. Conclusion: It was determined that, unlike other cationic reagents, the high transfection efficiency of HSA-PEI was not from the electronic effect but, instead, predominantly from its ligand effect.