Reduced-gliotoxin is a small molecule derived from the secondary metabolites of marine fungi; compared to other gliotoxin analogues, it exhibits potent anticancer effects. However, the molecular basis of the death of colorectal cancer (CRC) cells induced by reduced-gliotoxin is unclear. Thus, the aim of this study was to investigate the potency of reduced-gliotoxin against CRC cells and to elucidate the underlying mechanisms. Cell morphology, flow cytometric analysis and western bolt analysis were performed to examine the functions and mechanisms of cell death induced by reduced-gliotoxin. Our findings demonstrated that reduced-gliotoxin triggered rapid cell detachment and induced anoikis in CRC cells. Mechanistically, our data indicated that the anoikis induced by reduced-gliotoxin was associated with the disruption of integrin-associated cell detachment and multiple signaling pathways. Furthermore, reduced-gliotoxin induced the excessive production of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP), resulting in the activation of both endogenous and exogenous apoptotic pathways and eventually, in the apoptosis of CRC cells. The blockage of ROS generation with N-acetylcysteine (NAC) attenuated the anoikis induced by reduced-gliotoxin. Taken together, these results suggest that reduced-gliotoxin may prove to be a potential candidate in the treatment of CRC.