Herein, we have developed a mild and efficient synthesis of pyrazolo[3,4-e][1,4]thiazepine derivatives through an l-proline catalyzed multi-component reaction. The synthesized compounds were subjected to in vitro cytotoxicity studies toward pancreas (PANC1), renal (ACHN) and colon (HCT116), non-small cell lung (H460), lung (CALU1), and normal breast epithelium (MCF10A) cell lines and almost all the compounds were found to exhibit good to moderate activity. The compounds 4a, 4d, 4f, 4h, and 4k were found to possess prominent cytotoxic activity toward all cell lines tested. Of the compounds tested, 4f and 4k were found to possess activity equal to the control used toward all tested cell lines with 52.7-80.6 % GI values. © 2014 Springer Science+Business Media New York.