Zearalenone (ZEN) is a potent oestrogenic mycotoxin that is mainly produced by Fusarium species and is a serious environmental pollutant in animal feeds. Apiotrichum mycotoxinivorans has been widely used as a feed additive to detoxify ZEN. However, the effects of ZEN on A. mycotoxinivorans and its detoxification mechanisms remain unclear. In this study, transcriptomic and bioinformatic analyses were used to investigate the molecular responses of A. mycotoxinivorans to ZEN exposure and the genetic basis of ZEN detoxification. We detected 1424 significantly differentially expressed genes (DEGs), of which 446 were upregulated and 978 were downregulated. Functional and enrichment analyses showed that ZEN-induced genes were significantly associated with xenobiotic metabolism, oxidative stress response, and active transport systems. However, ZEN-inhibited genes were mainly related to cell division, cell cycle, and fungal development. Subsequently, bioinformatic analysis identified candidate ZEN-detoxification enzymes. The Baeyer–Villiger monooxygenases and carboxylesterases, which are responsible for the formation and subsequent hydrolysis of a new ZEN lactone, respectively, were significantly upregulated. In addition, the expression levels of genes related to conjugation and transport involved in the xenobiotic detoxification pathway were significantly upregulated. Moreover, the expression levels of genes encoding enzymatic antioxidants and those related to growth and apoptosis were significantly upregulated and downregulated, respectively, which made it possible for A. mycotoxinivorans to survive in a highly toxic environment and efficiently detoxify ZEN. This is the first systematic report of ZEN tolerance and detoxification in A. mycotoxinivorans. We identified the metabolic enzymes that were potentially involved in detoxifying ZEN in the GMU1709 strain and found that ZEN-induced transcriptional regulation of genes is key to withstanding highly toxic environments. Hence, our results provide valuable information for developing enzymatic detoxification systems or engineering this detoxification pathway in other species. •mycotoxinivorans GMU1709 strain can efficiently remove zearalenone (ZEN).•mycotoxinivorans possess complete xenobiotic detoxification pathways.•Exposure to ZEN would activate the antioxidant system of A. mycotoxinivorans.•Exposure to ZEN inhibit both the growth and apoptosis of A. mycotoxinivorans.