Lymphoid malignancies such as leukemia and many types of lymphoma are common and severe disorders in dogs. Since shortening remission duration caused by resistance to chemotherapy often becomes clinically critical problems, development of novel and effective therapy should be required. The present study investigated the status of NF-κB, and effect of its inhibitor, bortezomib, in six canine neoplastic lymphoid cell lines. NF-κB p65 and p50 were detected in the nuclear fraction of GL-1, CLBL-1 and CL-1, suggesting that NF-κB was constitutively activated in the cells. NF-κB p65 was detected in the cytoplasmic fraction of UL-1 and Ema. After incubation with bortezomib, NF-κB p50 and p65 became undetectable in the nuclear fraction of GL-1, CLBL-1 and CL-1, and CLBL-1, respectively, and p65 was clearly degraded in the cytoplasmic fraction of CLBL-1 and CL-1. Bortezomib inhibited the proliferation of all cell lines except Nody-1 in a concentration-dependent manner. The results indicated that constitutive activation of NF-κB could contribute to the proliferation of canine neoplastic lymphoid cells, and bortezomib would have suppressive effects on the NF-κB activation and the proliferation of neoplastic lymphoid cells in dogs.