We examined in vitro performance of the branched polyethylenimine (bPEI)-based gene carriers which respond to cancer-specific activation of protein kinase Cα (PKCα) to express plasmid DNA. The carriers were synthesized straightforward by using amide bond formation between a peptide terminal carboxyl and a primary amine group of bPEI. To examine the effect of the peptide contents in the carrier, we prepared several carriers with various peptide contents. The obtained polymers form polyplexes with tighter condensation of plasmid DNA than our previous gene carriers. After internalization of the polyplexes via endocytosis, the polyplexes effectively escaped from the endosome into cytosol. Then, the polyplexes showed a clear-cut response to PKCα to release plasmid DNA for gene expression. We determined the optimum contents of the peptides in carriers as 5 mol% to achieve the clear-cut response to PKCα.