<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Aims</jats:title> <jats:p>Cereals contaminated with type B trichothecene nivalenol (NIV) and its acetylated derivative 4-acetyl-nivalenol (4-AcNIV) are a global mycotoxicological problem threatening the health of humans and livestock. Toxicological studies, quantitative determinations and screening for biodegrading micro-organisms require massive amounts of pure toxins. However, the low yield from fungal cultures and high prices of NIV and 4-AcNIV limit research progress in these areas. This work aimed to select Fusarium asiaticum mutant strains with enhanced production of NIV and 4-AcNIV. </jats:sec> <jats:sec> <jats:title>Methods and Results</jats:title> <jats:p>A total of 62 NIV-producing F. asiaticum strains were isolated and compared regarding their ability to produce NIV. Strain RR108 had the highest yield of NIV among 62 field isolates surveyed and was then genetically modified for higher production. Targeted deletion of the FaFlbA gene, encoding a regulator of G protein signalling protein, resulted in a significant increase in NIV and 4-AcNIV production in the FaFlbA deletion mutant ΔFaFlbA. The expression of three TRI genes involved in the trichothecene biosynthetic pathway was upregulated in ΔFaFlbA. ΔFaFlbA produced the highest amount of NIV and 4-AcNIV when cultured in brown long-grain rice for 21 days, and the yields were 2.07 and 2.84 g kg−1, respectively. The mutant showed reduced fitness, including reduced conidiation, loss of perithecial development and decreased virulence on wheat heads, which makes it biologically safe for large-scale preparation and purification of NIV and 4-AcNIV. </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>The F. asiaticum mutant strain ΔFaFlbA presented improved production of NIV and 4-AcNIV with reduced fitness and virulence in plants. </jats:sec> <jats:sec> <jats:title>Significance and Impact of the Study</jats:title> <jats:p>Targeted deletion of the FaFlbA gene resulted in increased NIV and 4-AcNIV production. Our results provide a practical approach using genetic modification for large-scale mycotoxin production. </jats:sec>