The cytolethal distending toxin (Cdt) from Actinobacillus actinomycetemcomitans consists of three proteins, CdtA, CdtB, and CdtC, which are responsible for cell cycle arrest and apoptosis. In the present study, local delivery systems of recombinant CdtB and CdtB-expressing plasmid were established using Ca9-22, human gingival squamous cell carcinoma cell line. When CdtB was delivered to Ca9-22 cells using a BioPORTER, a 32-kDa protein was detected by Western blotting, and G2 cell cycle arrest and apoptosis occurred. In addition, the CdtB delivered upregulated the expression of phosphorylated p53 and the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in Ca9-22 cells, suggesting that these intracellular molecules might contribute to the induction of G2 cell cycle arrest and apoptosis. When the CdtB-expressing plasmid was transfected into Ca9-22 cells by lipofection or electroporation, CdtB (32 kDa) was clearly detected. Further, TdT-mediated dUTP nick end labeling positive cells were observed after transfection of the CdtB-expressing plasmid. These findings indicated that delivery of the CdtB protein and transfection of the cdtB gene induced cell cycle arrest and apoptosis in Ca9-22 cells in vitro, and we conclude that it may be possible to induce apoptosis in human gingival squamous cell carcinoma by electroporation of the cdtB gene.