Objective To investigate the mechanism of EFHD2 affecting the occurrence and progression of breast cancer based on NOX4/ROS signaling pathway. Methods The cells were divided into NC-shRNA group and EFHD2-shRNA group. The lentiviral vector silencing EFHD2 expression and its control vector were constructed and transfected MDA-MB-23 and MCF-7 breast cancer cells. The transfection efficiency was verified by qRT-PCR.CCK8 assay was used to detect cell proliferation activity. Plate cloning assay was used to detect cell colony formation ability. Scratch test was used to detect cell migration. Transwell assay was used to detect cell invasion. Flow cytometry was used to detect apoptosis and ROS levels. qRT-PCR was used to detect the mRNA expression of GLUT1, PDK1, PFK1, PKM2, PDH, and LDH. Western blot was used to detect the expression of Cleaved caspase-3, MMP-2 and NOX4 proteins. Results Compared with NC-shRNA group, EFHD2 expression was significantly decreased in EFHD2-SHRNA group, and cell survival and colony formation ability were weakened. The apoptosis rate and the expression of pro-apoptotic protein Cleaved caspase-3 increased. The cell migration distance was shortened, the number of cell invasion and the expression of MMP-2, which promotes migration and invasion, were decreased. The levels of lactic acid and GLUT1, PDK1, PFK1, PKM2 and LDH decreased, while the levels of ATP and PDH increased. Streaming results showed that ROS levels were reduced and NOX4 protein was down-regulated after silencing EFHD2. Conclusion EFHD2 can inhibit ROS production by regulating NOX4/ROS signaling pathway, cause lactic acid and glucose accumulation, promote apoptosis of breast cancer cells, and inhibit cell proliferation, migration and invasion.