Lycopene is a carotenoid mainly present in red-colored fruits and vegetables. Its value in the pharmaceutical and food industry is linked to its benefits for the human health, including properties against cancer and cardiovascular diseases, and its use as a food colorant. Lycopene can be produced either by synthetic or natural means, but there is a preference for the second, since it is considered a more eco-friendly and less harmful process. Among natural methods for obtaining lycopene, microbial fermentation is a good alternative to extraction from plants that naturally contain lycopene, since it implies obtaining higher and more specific amounts of this carotenoid. This chapter describes lycopene production by fermentation of the fungus Blakeslea trispora, a naturally carotenoid producer, at 30 L scale. This procedure involves separated growth of the two sexual mating types of B. trispora during the vegetative stages and the use of a lycopene cyclase inhibitor to achieve lycopene accumulation during the production stage.