Antitumor B (ATB), also known as Zeng Sheng Ping, is a Chinese herbal mixture composed of six plants. Previously, clinical studies have shown a significant chemopreventive efficacy of ATB against human esophageal and lung cancers. In the present study, A/J mice harboring a dominant-negative p53 and/or heterozygous deletion of Ink4a/Arf and treated with benzo[a]pyrene were used to investigate the chemopreventive effects of ATB on chemically induced lung tumorigenesis. Mice with various genotypes treated with ATB displayed a significant reduction in lung tumor multiplicity and tumor load. Treatment with ATB resulted in an approximately 40% decrease in tumor multiplicity and a 70% decrease in tumor load in both wild-type mice and in mice with a loss of the Ink4a/Arf tumor suppressor genes. Interestingly, ATB decreased tumor multiplicity and volume by 50 and 90%, respectively, in mice with a dominant-negative p53 and in mice with both a p53 mutation and deletion of Ink4a/Arf. Kras2 mutation analysis of the lung tumors revealed that tumors harbored mutations in the 12th codon of Kras2. There were no differences in either the incidence or types of mutations between tumors treated with or without ATB. Oligonucleotide array analysis revealed 284 genes that were differentially expressed in mouse lung tumors as compared to the normal lung, and it was found that 114 out of these 284 genes changed their expression toward the normal levels in tumors treated with ATB. Most of the genes modulated by ATB belong to several cellular signaling pathways, including Notch (Notch homolog 2, manic fringe homolog), growth factor (FGF intracellular-binding protein, PDGFalpha), G protein-Ras-MAPK (MAPK3, MAP3K4, rab3A, Rap1, RSG5, PKCtheta), ubiquitin-proteasome (CDC34, Cullin1, 26S proteasome), and apoptosis (BAD promoter, caspase 3). These results suggest that ATB is an effective chemopreventive against mouse lung tumorigenesis. Furthermore, ATB exhibited an enhanced inhibitory effect in animals harboring genetic alterations (Kras2, p53, and Ink4a/Arf), which are often seen in human lung adenocarcinomas.