Bacillus thuringiensis produces a variety of insecticidal crystal proteins (ICPs). Genome sequencing is a promising strategy for detecting and identifying B. thuringiensis ICPs, which are of great interest to the biocontrol field. In this study, a novel ICP gene was cloned from B. thuringiensis BRC-ZYR2 based on genomic data from 454 GS-FLX Titanium sequencing and an analysis of the results using the B. thuringiensis Toxin_Scanner ( http://bcam.hzaubmb.net/BtToxin_scanner/index.php). cry1Na3 designated by the B. thuringiensis Toxin Nomenclature Committee, encoded a 601-amino acid, 68.0-kDa protein that exhibited 95% identity with Cry1Na1 and 99% identity with Cry1Na2. Cry1Na3 contained three conserved domains commonly found in three-domain ICPs. Cry1Na3 was toxic to Plutella xylostella (L.) and Ostrinia furnacalis (Guenée), with LC₅₀ values of 3.69 µg/ml and 31.30 µg/ml, respectively. However, Laodelphax striatellus (Fallén) nymphs were unaffected when fed purified Cry1Na3 (250 µg/ml) in their diet. Spodoptera exigua (Hübner) and Colaphellus bowringi (Baly) larvae survived even when the concentration of Cry1Na3 protein reached 500 µg/ml. Cry1Na3 is a promising agent for the control of lepidopteran insect pests.