<p><i>Rhodotorula glutinis</i> is an important oleaginous yeast that can synthesize various valuable compounds, including carotenoids, lipids, and exopolysaccharides. The effect of combined heat stress and glucose starvation on carotenoid biosynthesis in <i>R. glutinis</i> was investigated in this study. Carotenoid production in <i>R. glutinis</i> was promoted by heat stress, and this effect was further enhanced when glucose starvation was applied to the strain. The results of multiomics analysis revealed that the effects of heat stress and glucose starvation on promoting carotenoid biosynthesis appeared to be additive, with the combined stress leading to a further increase in reactive oxygen species (ROS) levels and a reduction in enzymatic antioxidant capacity, while carotenoid biosynthesis was prioritized simultaneously. The key responses of <i>R. glutinis</i> to combined stress include the regulation of the cell cycle and energy metabolism, maintenance of membrane integrity, an increase in ROS scavenging capacity, and non-enzymatic antioxidant activity. Additionally, several candidate genes and metabolites associated with the combined stress response were identified. To summarize, we provided new insights into optimizing fermentation processes for increased carotenoid production in <i>Rhodotorula glutinis</i> and established a molecular basis for further genetic engineering to increase carotenoid yield.</p>