Superoxide　dismutase　(SOD)　fusion　of　TAT　was　proved　to　be　radioprotective　in　our　previous　work.　On　that　basis,　a　bifunctional　recombinant　protein　which　was　the　fusion　of　glutathione　S-transferase　(GST),　SOD,　and　TAT　was　constructed　and　named　GST-TAT-SOD.　Herein　we　report　the　investigation　of　the　cytotoxicity,　cell-penetrating　activity,　and　in　vitro　radioprotective　effect　of　GST-TAT-SOD　compared　with　wild　SOD,　single-function　recombinant　protein　SOD-TAT,　and　amifostine.　We　demonstrated　that　wild　SOD　had　little　radioprotective　effect　on　irradiated　L-02　and　Hep　G2　cells　while　amifostine　was　protective　to　both　cell　lines.　SOD-TAT　or　GST-TAT-SOD　pretreatment　3　h　prior　to　radiation　protects　irradiated　normal　liver　cells　against　radiation　damage　by　eliminating　intracellular　excrescent　superoxide,　reducing　cellular　MDA　level,　enhancing　cellular　antioxidant　ability　and　colony　formation　ability,　and　reducing　apoptosis　rate.　Compared　with　SOD-TAT,　GST-TAT-SOD　was　proved　to　have　better　protective　effect　on　irradiated　normal　liver　cells　and　minimal　effect　on　irradiated　hepatoma　cells.　Besides,　GST-TAT-SOD　was　safe　for　normal　cells　and　effectively　transduced　into　different　organs　in　mice,　including　the　brain.　The　characteristics　of　this　protein　suggest　that　it　may　be　a　potential　radioprotective　agent　in　cancer　therapy　better　than　amifostine.　Fusion　of　two　antioxidant　enzymes　and　cell-penetrating　peptides　is　potentially　valuable　in　the　development　of　radioprotective　agent.　©　2016　Jianru　Pan　et　al.