Several studies have reported that plant-derived natural products have cancer chemopreventive and chemotherapeutic properties. The aim of the present study was to determine the antiproliferative and pro-apoptotic potential of Limoniastrum guyonianum aqueous gall extract (G extract) on human colorectal cancer BE cell line and, if so, to characterize the mechanism involved. The G extract-induced growth inhibitory effect was associated with an arrest of cell cycle progression in G2/M phase as shown by the cell phase distribution. In addition, G extract promoted in a concentration-dependent manner these cells towards apoptosis as indicated by the presence of cleaved poly(ADP-ribose) polymerase (PARP). In order to characterize the mechanism involved in the antiproliferative and pro-apoptotic signaling pathway activated by G extract, calpain activity and the expression of the cell cycle inhibitor p16(INK4A) were determined. The present findings indicated that G extract exhibited significant inhibitory activity against calpain and caused a marked and concentration-dependent upregulation of p16(INK4A). These effects could be ascribed to the presence of condensed tannins and polyphenols such as epicatechin and epigallocatechin gallate in G extract.