The co-culturing of Pleurotus eryngii var. ferulae and Rhodotorula mucilaginosa was confirmed in our previous studies to be an efficient strategy to improve laccase production by submerged fermentation. To determine the possible regulation principles underlying this behaviour, comparative transcriptomic analysis was performed on P. eryngii var. ferulae to investigate the differential expression of genes in co-culture. RNA-seq analysis showed that genes concerning xenobiotic biodegradation and expenditure of energy were upregulated. However, genes related to oxidative stress were downregulated. In addition, the transcription levels of laccase isoenzymes were not consistent in the co-culture system: 3 laccase genes (lacc1, lacc2, lacc12) were upregulated, and 3 laccase genes (lacc4, lacc6, lacc9) were downregulated. The enhancement in laccase activity can be due to upregulation of a laccase heterodimer encoded by the genes lacc2 and ssPOXA3a (or ssPOXA3b), whose expression levels were increased by 459% and 769% (or 585% for ssPOXA3b) compared with those of a control, respectively. β-Carotene produced by R. mucilaginosa upregulated the transcription of lacc2 only. Combining these results with an analysis of cis-acting responsive elements indicated that four transcription factors (TFs) had potential regulatory effects on the transcription of laccase genes. It was supposed that TFa regulated lacc transcription by binding with methyl jasmonate and heat shock response elements. The expression of TFb, TFc, and TFd was regulated by β-carotene. However, β-carotene had no effect on TFa expression. These results provide a possible mechanism for the regulation of laccase gene transcription in the co-culture system and are also beneficial for the future intensification of fungal laccase production.