The 70-kD heat shock proteins (Hsp70s) have been proved to be important for stress tolerance and protein folding and unfolding in almost all organisms. However, the functions of Hsp70s in mushroom are not well understood. In the present study, a hsp70 gene from Hypsizygus marmoreus, hmhsp70, was cloned and transferred to tobacco (Nicotiana tabacum) to evaluate its function in thermotolerance. Sequence alignments and phylogenetic analysis revealed that HmHsp70 may be located in the mitochondria region. qPCR analysis revealed that the transcription level of hmhsp70 in H. marmoreus mycelia increased after heat shock treatment in high temperature (42 °C) compared with untreated mycelia (at 25 °C). Transgenic tobaccos expressing hmhsp70 gene showed enhanced resistance to lethal temperature compared with the wild type (WT) plants. Nearly 30% of the transgenic tobaccos survived after treated at a high temperature (50 °C and 52 °C for 4 h); however, almost all the WT tobaccos died after treated at 50 °C and no WT tobacco survived after heat shock at 52 °C. This study firstly showed the function of a hsp70 gene from H. marmoreus.