Endophytic fungi are an important source of novel antitumour substances. Previously, we isolated an endophytic fungus, Alternaria alstroemeria, from the medicinal plant Artemisia artemisia, whose crude extracts strongly inhibited A549 tumour cells. We obtained a transformant, namely AaLaeA^OE26, which completely loses its antitumour activity due to overexpression of the global regulator AaLaeA. Re-sequencing analysis of the genome revealed that the insertion site was in the non-coding region and did not destroy any other genes. Metabolomics analysis revealed that the level of secondary antitumour metabolic substances was significantly lower in AaLaeA^OE26 compared to the wild strain, in particular flavonoids were more downregulated according to the metabolomics analysis. A further comparative transcriptome analysis revealed that a gene encoding FAD -binding domain protein (Fla1) was significantly downregulated. On the other hand, overexpression of AaFla1 led to significant enhancement of antitumor activity against A549 with a 7-fold higher inhibition ratio than the wild strain. At the same time, we also found a significant increase in the accumulation of antitumour metabolites including quercetin, gitogenin, rhodioloside, liensinin, ginsenoside Rg2 and cinobufagin. Our data suggest that the global regulator AaLaeA negatively affects the production of antitumour compounds via controlling the transcription of AaFla1 in endophytic Alternaria alstroemeria.