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A white rot fungus <i>Cerrena unicolor</i> has been identified as an important source of laccase, unfortunately regulation of this enzyme genes expression is poorly understood. Using 1D and 2D PAGE and LC-MS/MS, laccase isoenzymes were investigated in the liquid filtrate of <i>C. unicolor</i> culture. The level of expression of laccase genes was measured using qPCR. The elevated concentrations of copper and manganese in the medium caused greatest change in genes expression and three laccase transcripts were significantly affected after culture temperature was decreased from 28 to 4 °C or increased to 40 °C. The small differences in the PAGE band intensities of individual laccase proteins were also observed, indicating that given compound affect particular laccase’s transcript. Analyses of laccase-specific activity, at all tested conditions, showed the increased activities as compared to the control, suggesting that enzyme is regulated at the post-translational stage. We observed that the aspartic protease purified from <i>C. unicolor</i>, significantly stimulate laccase activity. Moreover, electrochemical analysis of protease-treated laccase sample had 5 times higher redox peaks. The obtained results indicate that laccases released by <i>C. unicolor</i> are regulated at transcriptional, translational, and at the post-translational steps of gene expression helping fungus adapt to the environmental changes.