Crosslinked bioreducible polypropylenimine-cystaminebisacrylamides (PPI-CBAs) were synthesized for gene delivery systems. They formed nano-sized polyplexes with high stability even in reducing condition probably due to the re-crosslinking. PPI-CBAs displayed high transfection efficiency comparable to PEI25k in serum condition. However, cytotoxicity increase, glutathione (GSH) decrease, and reactive oxygen species (ROS) increase were observed in cells with the elevation of PPI-CBAs concentration and crosslinking degree. These results suggest that the cytotoxicity of bioreducible polymers may be closely related with their structures and that reduction of GSH by degradation of re-crosslinked bioreducible polymers and the following increase of ROS may induce cytotoxicity by oxidative stress.