Overexpressed TBC1D8B, a GTPase-activating protein, significantly reduced cultured HCT116 human colon cancer cell number. We tested N-terminal TBC1D8B, which is identical to wild type TBC1D8B from amino acid positions 1 to 427 and possesses a modified sequence from position 428 to 435 (ECGGLFLL) because of the introduction of a premature stop codon at position 436 to narrow down the minimum requirement element. The N-terminal TBC1D8B contains two GRAM domains but not the TBC domain essential for Rab-GTPase activity. The N-terminal TBC1D8B overexpression significantly reduced the cultured HCT116 cell number. When we tested C-terminal TBC1D8B, containing the portion of TBC1D8B absent in the N-terminal TBC1D8B, the cell number reduction was not observed. The N-terminal TBC1D8B overexpression significantly increased the coronin 1B expression and reduced the phosphorylation of serine 51 in eIF2α, respective markers of apoptosis and cell death/survival. Also, caspase 3 and poly ADP-ribose polymerase increased cleavage in suspended cells overexpressing the N-terminal TBC1D8B. Taken together, it is not the TBC domain for Rab-GTPase activity, but amino acids 1 to 435, including the two GRAM domains, that is enough for TBC1D8B to cause spontaneous apoptosis. TBC1D8B could be a potential anticancer therapeutic molecule.