The exploitation of symbiotic interactions between fungi and plants, coupled with the application of osmoprotectants such as trehalose (Tre), presents a promising strategy for mitigating environmental stress. To determine the mechanism of Serendipita indica and Tre-mediated cold stress tolerance, a comparative experiment was designed to study the impact of S. indica, Tre and their combination on tomato plants grown under cold stress. The results showed that cold stress significantly decreased biomass, relative water content, photosynthetic pigments and elements concomitantly with increasing antioxidant activities, malondialdehyde (MDA), electrolyte leakage, hydrogen peroxide and proline content. Meanwhile, S. indica and Tre treatments promoted biomass and enhanced carbohydrate, protein, proline, potassium, phosphorous, antioxidant enzymes and photosynthetic pigments content under cold stress. Furthermore, single or dual application of endophyte and Tre mitigated physiological disorders induced by cold stress and increased the integrity of cell membranes by decreasing hydrogen peroxide, MDA, and electrolyte leakage (EL). Our findings suggest that S. indica and Tre combination could significantly promote cold stress tolerance compared with single treatment. This study is novel in showing the cold adaptation of tomato plants by combination use of S. indica and Tre, which can be a promising strategy for improving cold tolerance. The underlying molecular mechanisms of sugar–fungus interaction must be further investigated.