Human GGCT (γ-glutamyl cyclotransferase) has been shown to be upregulated in most tumors, but its role in colorectal cancer (CRC) is poorly understood. Thus, CRC cell lines, including HCT116 and SW1116, were chosen to investigate the role of GGCT by constructing a GGCT silencing cells model using lentivirus-mediated RNA interference. The knockdown efficiency was confirmed by reverse transcription-quantitative PCR and a western blot assay. Then, a set of biological functions of GGCT silencing on CRC cell was assessed by MTT, colony-formation assay, and flow cytometry analysis. Further, western blot and Pathscan intracellular signaling were used to detect intracellular signaling associated with cell growth and apoptosis induced by GGCT knockdown. In addition, the clinical chemotherapeutic drug 5-fluorouracil was used to investigate the impact of GGCT silencing on drug sensitivity by an Annexin V/7-AAD double-staining assay. The results of the analysis indicated that GGCT silencing significantly suppressed cell proliferation and arrested cell cycle at the G0/G1 phase by regulating the expression of p21, p27, and cyclin E. Moreover, GGCT silencing triggered the apoptosis of CRC cells by activating caspase-3 and cleaved poly-ADP-ribose polymerase pathways and downregulating the phosphorylation proline-rich Akt substrate of 40 kDa (PRAS40) expression levels. Furthermore, GGCT silencing combined with 5-fluorouracil treatment further induced the apoptotic rate of CRC cells. These findings suggest that GGCT may be a promising diagnostic and therapeutic target for CRC by activating the apoptotic pathway.