Both cDNA and a genomic DNA fragment encoding a new potato proteinase inhibitor II were isolated from a diploid potato IVP101 (Solanum phurejia L.) and named PINII-2x. Nucleotide sequencing confirmed that the DNA fragment of PINII-2xwas 580 bp, including a 115-bp intron and two exons. The deduced PINII-2x protein contained an intact signal peptide and two active sites. The PINII-2x gene and its deduced PINII-2x protein had 88% and 93% homology with another tetraploid potato proteinase inhibitor II, respectively. Northern blotting analysis indicated that the mRNA of PINII-2x gene was wound induced in potato leaves. Binary vector pNAR301 and pNAR302 were constructed for rice transformation, in which the PINII-2x cDNA was driven, respectively, by rice actin I promoter (ActI) and maize ubiquitin promoter (UbiI). Via an Agrobacterium-mediated method, these two constructs were transferred into japonica rice cv. Xiushui 63. PCR and Southern blotting analysis for transgenic rice revealed the integration of the PINII-2x gene. Northern blotting analysis also confirmed transcripts of the PINII-2x gene in transgenic rice plants. Insect bioassays using stripe stem borer (Chilo suppressalis Walker) demonstrated that the average weight and body length of larvae in transgenic plants were only nearly 50% and 61% of those of larvae in control plants, respectively. These results indicate that the PINII-2x gene should be an effective insect-resistance gene and could be valuable for application in crop breeding for insect resistance.