IntroductionFlammulina filiformisis one of the most commercially important edible fungi worldwide, with its nutritional value and medicinal properties. It becomes a good model species to study the tolerance of abiotic stress during mycelia growth in edible mushroom cultivation. Transcription factor Ste12 has been reported to be involved in the regulation of stress tolerance and sexual reproduction in fungi.MethodsIn this study, identification and phylogenetic analysis ofste12-likewas performed by bioinformatics methods. Fourste12-likeoverexpression transformants ofF. filiformiswere constructed by Agrobacteriumtumefaciens-mediated transformation.Results and DiscussionPhylogenetic analysis showed that Ste12-like contained conserved amino acid sequences. All the overexpression transformants were more tolerant to salt stress, cold stress and oxidative stress than wild-type strains. In the fruiting experiment, the number of fruiting bodies of overexpression transformants increased compared with wild-type strains, but the growth rate of stipes slowed down. It suggested that geneste12-likewas involved in the regulation of abiotic stress tolerance and fruiting body development inF. filiformis.