The growth inhibition and cytotoxic effects of theaflavins mixture and pure theaflavins[theaflavin, theaflavins-3-gallate, theaflavins-3′-gallate and theaflavin-3,3′-digallate] from black tea on human liver cancer HepG2 cells, gastric cancer SGC-7901 cells, lung cancer A549 cells and acute promyelocytic leukemia K562 cells were investigated. The four major theaflavins were obtained fromenzymatically oxidized catechins mixture using preparative high-performance liquid chromatography (HPLC).The purity and the structures of the isolated compoundswere confirmed by 1H nuclear magnetic resonance, LC/MS, Fourier-transform infrared, HPLC with diode array detection and thin-layer chromatography analyses. Theaflavin monomers and mixture exhibited strong inhibitory effects on SGC-7901 and A549 cells.The obtained TF2A exhibited high inhibition activity against the growth of all four cancer cells examined. It is concluded that individual theaflavins have differential inhibitory activity incancer cell linesandthatTF2Ahave promisingactivity tobeconsidered in anticancer treatment research as a drug or a template for drug synthesis.