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The protein encoded by immature colon carcinoma transcript 1 (ICT1) is a component of the human mitochondrial ribosome, and is reported to be implicated in cell proliferation, viability and apoptosis of HeLa cells. This study was conducted to investigate the role of ICT1 in human breast cancer. Oncomine database was used to investigate ICT1 expression in human breast cancer tissues compared to normal tissues. The results showed that ICT1 was highly overexpressed in various human breast cancer subtypes. Then short hairpin RNA (shRNA)-mediated knockdown of ICT1 was performed in human breast cancer ZR-75-30 and T-47D cells. A series of functional analysis, including MTT, colony formation and flow cytometry assays were conducted after ICT1 knockdown. Our results demonstrated that knockdown of ICT1 significantly suppressed cell viability and proliferation through cell cycle arrest at the G2/M phase and induced apoptosis in breast cancer cells. Furthermore, knockdown of ICT1 altered signaling pathways associated with cell growth and apoptosis, including phospho‑BAD (Ser112), phospho-PRAS40 (Thr246) and induction of phospho‑AMPKα (Thr172). Additionally, it was further confirmed by western blot analysis that ICT1 knockdown altered the expression of apoptosis- or cell cycle‑related proteins such as Bcl-2, caspase-3, CDK1, CDK2 and cyclin B. In conclusion, targeting ICT1 in breast cancer cells may provide a new strategy for breast cancer gene therapy.