Dimethylaminoparthenolide (DMAPT) is a water soluble parthenolide analog with preclinical activity in hematologic malignancies. Using non–small lung cancer (NSCLC) cell lines (A549 and H522) and an immortalized human bronchial epithelial cell line (BEAS2B) and TCC cell lines (UMUC-3, HT-1197 and HT-1376) and a bladder papilloma (RT-4), we aimed to characterize DMAPT's anticancer activity in tobacco-associated neoplasms. Flow cytometric, electrophoretic mobility gel shift assays (EMSA), and Western blot studies measured generation of reactive oxygen species (ROS), inhibition of NFκB DNA binding, and changes in cell cycle distribution and apoptotic proteins. DMAPT generated ROS with subsequent JNK activation and also decreased NFκB DNA binding and antiapoptotic proteins, TRAF-2 and XIAP. DMAPT-induced apoptotic cell death and altered cell cycle distribution with upregulation of p21 and p73 levels in a cell type–dependent manner. DMAPT suppressed cyclin D1 in BEAS2B. DMAPT retained NFκB and cell cycle inhibitory activity in the presence of the tobacco carcinogen nitrosamine ketone, 4(methylnitrosamino)-1-(3–pyridyl)-1-butanone (NNK). Using a BrdU accumulation assay, 5–20 μM of DMAPT was shown to inhibit cellular proliferation of all cell lines by more than 95%. Oral dosing of DMAPT suppressed in vivo A549 and UMUC-3 subcutaneous xenograft growth by 54% (p = 0.015) and 63% (p < 0.01), respectively, and A549 lung metastatic volume by 28% (p = 0.043). In total, this data demonstrates DMAPT's novel anticancer properties in both early and late stage tobacco-associated neoplasms as well as its significant in vivo activity. The data provides support for the conduct of clinical trials in TCC and NSCLC.