Abstract: Lycopene cyclase/β-hydroxy-lycopene synthase (CrtYB) is a key enzyme involved in carotenoid biosynthesis. In this study, we cloned the CrtYB gene from the Rhodosporidium kratochvilovae YM25235 strain and designated it RkCrtYB. The full-length RkCrtYB gene is 1 785 bp and encodes 594 amino acids. Sequence analysis of the RkCrtYB gene revealed a high degree of similarity to known CrtYB sequences. Functional validation further showed that overexpression of RkCrtYB in YM25235 resulted in a 35.98% increase in carotenoid content, while the lipid content also increased by approximately 17.32%. This phenomenon is likely due to the increased synthesis of carotenoids, which requires the accumulation of additional lipid droplets for storage, thereby promoting lipid accumulation. Gene expression analysis supported this hypothesis. Collectively, our findings demonstrate that overexpression of RkCrtYB in YM25235 significantly enhances both carotenoid biosynthesis and lipid accumulation. This study provides new insights and a reference for the development of oil-producing strains with high carotenoid yields through metabolic engineering. To our knowledge, similar studies have not been reported in oil-producing red yeasts.