The nematicidal activity of 92 Bacillus thuringiensis (Bt) strains against the pine wood nematode Bursaphelenchus xylophilus, one of the world's top 10 plant-parasitic nematodes, was determined. The insecticidal crystal proteins (ICPs) from Bt BRC-XQ12 were the most toxic to B. xylophilus with an LC50 of 32.13 μg/mL. Since the ICPs expressed by Bt BRC-XQ12 were closest to Cry1Ea6 and Bt BRC-XQ12 contained four kinds of cry1 subgenes (cry1Aa, cry1Cb, cry1Ea and cry1Ia), Cry1Ea was most likely to be the key active component against the nematode. The 3516 bp cry1Ea11 gene from Bt BRC-XQ12, as designated by the Bt delta-endotoxin nomenclature committee, was expressed in Escherichia coli. Purified Cry1Ea11 showed an LC50 of 32.53 μg/mL and 23.23 μg/mL at 24 h and 48 h with corresponding virulence equations of Y=32.15X + 1.38 (R(2)=0.9951) and Y=34.29X + 3.16 (R(2)=0.9792), respectively. In order to detect the pathway of Bt Cry1Ea11 entering into B. xylophilus, the nematode was fed with NHS-rhodamine-labeled GST-Cry1Ea11. The results of confocal laser scanning microscopy (CLSM) showed that the 159 kDa GST-Cry1Ea11 could be detected in the stylet and the esophageal lumen of the pine wood nematode, indicating that GST-Cry1Ea11 could enter into the nematode through the stylet. As far as we know, no Cry1 proteins have activity against plant-parasitic nematodes before. These results demonstrate that Cry1Ea11 is a promising nematicidal protein for controlling pine wilt disease (PWD) rendered by B. xylophilus, further dramatically broadening the spectrum of Bt ICPs.