Gene therapy through systemic administration is expected to offer significant therapeutic potential against intractable cancers, including pancreatic cancer. One of the requirements for in vivo gene therapy is the development of a gene carrier with a high level of safety, transfection ability and tumour accumulation. Bovine serum albumin (BSA)-poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) conjugation (BSA-PDMAEMA conjugation) could result in the development of a promising gene carrier. This conjugate could preserve the BSA structure well and efficiently condense the nucleotide inside, resulting in the formation of BSA-PDMAEMA nanoparticles that have a polyion complex core and surrounding BSA corona with a size of <100 nm. The nanoparticles that were produced based on BSA-PDMAEMA conjugation possessed good characteristics for use as a gene carrier with good biocompatibility, appropriate blood retention and gene protective properties. Furthermore, the in vivo two-dimensional and three-dimensional biodistribution in a xenograft pancreatic cancer (AsPC-1) model in mice clearly showed that BSA-PDMAEMA nanoparticles accumulated at the tumour site via enhanced permeability and the retention effect. Furthermore, BSA-PDMAEMA nanoparticles, which condensed the active anti-cancer oligonucleotide, ISIS5132, inhibited the growth of cancer in AsPC-1-bearing mice compared to mice which were administered with ISIS5132 alone. The structure of the BSA-PDMAEMA nanoparticles, i.e. the polyion complex core with the BSA corona, would comprehensively contribute to these ideal characteristics for use as a gene carrier. In conclusion, BSA-PDMAEMA nanoparticles could exert a therapeutic effect on intractable pancreatic cancer in vivo, indicating their use as a promising gene carrier.