In order to explore the effects of low temperature stress on intestinal oxidative stress, apoptosis and intestinal microbial composition of Takifugu fasciatus, experiments were conducted to measure oxidative stress, apoptosis and intestinal microbe-related indicators at 3 treatment temperatures (25, 19 and 13 °C) and 4 sampling time periods (0, 6, 24 and 96 h). Compared with the control group, the results showed that as the temperature decreased, intestinal villi underwent necrosis, hyperplasia and enlargement of goblet cells, the enzymatic activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) increased significantly, while the diversity of gut microbial composition decreased. Changes in gut microbial abundance, such as Xanthobacter, Paracocuus, Lactococcus, and Bididobacter increased with decreasing temperature, Clostridium, Aeromonas and Geobacillus decreased with increasing temperature, and the expression of apoptosis-related genes (caspase3, caspase7, caspase9, p53, Bax, Bcl-2) increased with the decrease of temperature. In addition, we found that the alterations in the abundance of Paracocuus and Geobacillus had Pearson correlations with intestinal oxidative stress and apoptosis, which may be potential microbes to reflect whether the T. fasciatus is in a state of oxidative stress and apoptosis. The results of this study provide reference for the healthy breeding of T. fasciatus.