Thymosin β(4) (Tβ(4)), overexpressed in various tumors, has been shown to be involved in cellular anti-oxidation. Reactive oxygen species (ROS) function as signaling molecules and play certain roles in tumor progression. To assess the anti-oxidative role of endogenous Tβ(4) in tumor cells, its expression in SW480 cells was knocked down by a shRNA, which induced significant increases of ROS. Interestingly, some cristae-lost and several electron-dense mitochondria appeared in cells with Tβ(4) knockdown that was accompanied by a marked decline of the membrane potential of these organelles. Strikingly, while the ATP and lactate levels in SW480 cells were notably elevated by Tβ(4) downregulation, this treatment significantly diminished the mitochondrial DNA copy number and protein levels of several subunits of the electron transport complexes. Finally, immunofluorescent staining results suggested the presence of Tβ(4) in mitochondria. To the best of our knowledge, this is the first report to demonstrate that Tβ(4) knockdown can disrupt the morphology and some crucial functions of mitochondria in human colorectal carcinoma (CRC) cells.