Neurosporaxanthin is a carotenoid with a carboxylic moiety, produced by some fungi such as those from the genera Neurospora and Fusarium, which possesses strong antioxidant activity and is a source of vitamin A. The fungus Fusarium fujikuroi has been the subject of detailed research on the genes involved in neurosporaxanthin biosynthesis and its regulatory mechanisms, which resulted in the discovery of the key repressor gene carS. The ease of identifying carotenoid-overproducing mutants affected in this gene in some Fusarium species has been complemented by the recent development of culture media for these mutants, which allow the accumulation of this carotenoid in large quantities, as well as appropriate HPLC separation conditions for its analysis and purification. This chapter describes the methodology available to produce and purify extracts rich in neurosporaxanthin, from using a simple mutagenesis technique to obtain suitable overproducing strains to generating the final extract, including its further purification, thus facilitating the potential industrial application of these neurosporaxanthin-rich extracts as food additives, nutraceuticals, or cosmetics.