Canthaxanthin is a natural diketo derivative of β-carotene primarily used by the food and feed industries. Mucor circinelloides is a β-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the β-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and L-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 μg/g (dry mass) of canthaxanthin.