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<i>Pleurotus ostreatus</i> is a typical tetrapolar heterologous edible mushroom, and its growth and development regulatory mechanism has become a research hotspot in recent years. The MAC1 protein is a transcription factor that perceives copper and can regulate the expression of multiple genes, thereby affecting the growth and development of organisms. However, its function in edible mushrooms is still unknown. In this study, two transcription factor genes, <i>PoMCA1a</i> and <i>PoMAC1b,</i> were identified. Afterwards, <i>PoMAC1</i> overexpression (OE) and RNA interference (RNAi) strains were constructed to further explore gene function. The results showed that the <i>PoMAC1</i> mutation had no significant effect on the growth rate of mycelia. Further research has shown that OE-<i>PoMAC1a</i> strains and RNAi-<i>PoMAC1b</i> strains exhibit strong tolerance under 32 °C heat stress. However, under 40 °C heat stress, the OE of <i>PoMAC1a</i> and <i>PoMAC1b</i> promoted the recovery of mycelial growth after heat stress. Second, the OE of <i>PoMAC1a</i> can promote the rapid formation of primordia and shorten the cultivation cycle. In summary, this study indicated that there are functional differences between <i>PoMAC1a</i> and <i>PoMAC1b</i> under different heat stresses during the vegetative growth stage, and <i>PoMAC1a</i> has a positive regulatory effect on the formation of primordia during the reproductive growth stage.