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White-rot fungi, especially Trametes strains, are the primary source of industrial laccases in bioenergy and bioremediation. Trametes strains express members of the laccase gene family with different physicochemical properties and expression patterns. However, the literature on the expression pattern of the laccase gene family in Trametes trogii S0301 and the response mechanism to Cu(2+), a key laccase inducer, in white-rot fungal strains is scarce. In the present study, we found that Cu(2+) could induce the mRNAs and proteins of the two alternative splicing variants of heat shock transcription factor 2 (TtHSF2). Furthermore, the overexpression of alternative splicing variants TtHSF2α and TtHSF2β-I in the homokaryotic T. trogii S0301 strain showed opposite effects on the extracellular total laccase activity, with maximum laccase activities of approximately 0.6 and 3.0 U ml(−1), respectively, on day 8, which are 0.4 and 2.3 times that of the wild-type strain. Similarly, TtHSF2α and TtHSF2β-I play opposite roles in the oxidation tolerance to H(2)O(2). In addition, the direct binding of TtHSF2α to the promoter regions of the representative laccase isoenzymes (TtLac1 and TtLac13) and protein-protein interactions between TtHSF2α and TtHSF2β-I were detected. Our results demonstrate the crucial roles of TtHSF2 and its alternative splicing variants in response to Cu(2+). We believe that these findings will deepen our understanding of alternative splicing of heat shock transcription factors (HSFs) and their regulatory mechanism of the laccase gene family in white-rot fungi. IMPORTANCE The members of laccase gene family in Trametes strains are the primary source of industrial laccase and have gained widespread attention. Increasing the yield and enzymatic properties of laccase through various methods has always been a topic worthy of attention, and there is no report on the regulation of laccase expression through HSF transcription factor engineering. Here, we found that two alternative splicing variants of TtHSF2 functioned oppositely in regulating the expression of laccase genes, and copper can induce the expression of almost all members of the laccase gene family. Most importantly, our study suggested that TtHSF2 and its alternative splicing variants are vital for copper-induced production of laccases in T. trogii S0301.