The mechanism of the low temperature autolysis of Volvariella volvacea (V. volvacea) has not been thoroughly explained, and trehalose is one of the most important osmolytes in the resistance of fungi to adversity. The present study used the low temperature sensitive V. volvacea strain V23 and the low temperature tolerant strain VH3 as test materials. Intracellular trehalose contents under low temperature stress in the two strains were measured by high performance liquid chromatography (HPLC). Quantitative real-time PCR (qPCR) analysis was carried out to study the transcriptional expression differences of enzymes related to trehalose metabolism. And trehalose solution was exogenously added during the cultivation of fruit bodies of V. volvacea. The effect of exogenous trehalose solution on the anti-hypothermia of fruit bodies was studied by evaluating the sensory changes under low temperature storage after harvest. The results showed that the intracellular trehalose content in VH3 was higher than that in V23 under low temperature stress. In the first 2 h of low temperature stress, the expression of trehalose-6-phosphate phosphatase (TPP) gene involved in trehalose synthesis decreased, while the expression of trehalose phosphorylase (TP) gene increased. The expression of TPP gene was almost unchanged in VH3, but it decreased dramatically in V23 at 4 h of low temperature stress. The expression levels of TPP and TP genes in VH3 was significantly higher than that in V23 from 6 h to 8 h of low temperature stress. TP gene may be a crucial gene of trehalose metabolism, which was more inclined to synthesize trehalose during low temperature stress. In addition, the sensory traits of V. volvacea fruit bodies stored at 4 °C were significantly improved by the application of exogenous trehalose compared with the controls. Thus, trehalose could help V. volvacea in response to low temperature stress and high content of it may be one of the reasons that why VH3 strain was more tolerant to the low temperature stress than V23 strain.