Objective To prepare antibody-targeting temperature-sensitive polylated N-isopropylacrylamide/polyethyleneimine (PNIPAM/PEI) nanogel for delivering siRNA against ribonucleotide reductase (RR) subunit M2 (RRM2, RRM2-siRNA) and to establish a new targeted nano-gel delivery system for anti-tumor therapy by studying its anti-tumor ability in vitro. Methods We synthesized the core-shell temperature-sensitive PNIPAM/PEI nanogel by radical graft copolymerization and evaluated its chemo-physical properties (such as, size and zeta potential) using transmission electron microscopy (TEM). According to the principle of charge interaction, the PNIPAM/PEI-siRNA nanogel encapsulated RRM2-siRNA was prepared by conjugating anti-human epidermal growth factor receptor 2 (Her2) antibody. The effect of PNIPAM/PEI-siRNA nanogel complex encapsulated siRNA was determined by agarose gel electrophoresis. The in vitro uptake of NCI-N87 cells by nanogel complex was quantitatively observed by fluorescence microscopy and flow cytometry (FCM). The expression of RRM2 after RRM2 interference using nanogel-siRNA complex in NCI-N87 cells was tested by realtime PCR. The tumor migration suppressing effect of the nanogel complex on Her2-positive tumor cells was determined by Transwell assay. Results The core-shell temperature-responsive PNIPAM/PEI nanogel was synthesized by radical graft copolymerization, with homogeneous size of 359.8 nm, and zeta potential of 21.4 mV. Furthermore, the nanogel complexes encapsulated RRM2-siRNA with different ratios of N/P (N/P ratio: the molar ratio of nitrogen-from-polyethylenimine to phosphate-from-RNA) were prepared and the electrophoresis results showed that the optimal N/P ratio was 60. The cellular uptake experiment showed that the nanogel had good temperature sensitivity and tumor targeting ability at different temperatures (37 ℃, 42 ℃); and the nanogel complex down-regulated the expression of RRM2 and inhibited the migration of NCI-N87 cells. Conclusion The antibody-targeting temperature-sensitive PNIPAM/PEI nanogel is successfully prepared, and it can bind and deliver siRNA into target cells and can be used as a new drug delivery system for anti-tumor therapy.