Kaempferia rotunda Linn. is a plant of ginger family and has many medicinal values in traditional applications, including as antimicrobial and anticancer agents. The present study aims to examine the anticancer activity of Kaempferia rotunda tuberous rhizome lectin (KRL, MW 29 ± 1.0 kDa) against colon cancer cells SW480 and SW48. KRL inhibited 67% and 59% of SW480 and SW48 cells growth respectively at the concentration of 1.0 mg/mL. The cells growth inhibition was a dose dependent manner. KRL treatments notably inhibited the colony formation capacity of the cancer cells. The surviving fractions of SW480 and SW48 cells treated with KRL significantly (p < 0.001) reduced compared to that of control cells. Significant increment of the apoptotic cells were noted following by G0/G1 or G2/M cell cycle arrest in KRL treated SW480 and SW48 cells, respectively. Modulation of PARP1, p53, p21, Bax and Bcl2 proteins expression was observed in treated cells in comparison to that of untreated cells. Furthermore, activation of caspase-3 and caspase-9 was noted in KRL treated cells and caspase-3 and caspase-9 inhibitors pre-treated cells were shown insensitive to KRL treatment. The results implied that KRL prevents SW480 and SW48 cells proliferation by the induction of apoptosis in the mitochondrial intrinsic pathway.