Background <p>Vitamin A, an essential micronutrient, is critical for vision, immune function, and cellular differentiation. Traditional vitamin A production methods, primarily based on chemical synthesis, pose significant environmental challenges. Microbial fermentation offers a sustainable alternative, but microbial production of vitamin A has yet to match chemical synthesis in terms of yield and cost-effectiveness. Oleaginous yeasts, such as <i>Lipomyces starkeyi</i>, which can synthesize high levels of acetyl-CoA and lipids, represent promising platforms for producing high-value lipophilic compounds, such as vitamin A.</p> Results <p><i>L. starkeyi</i> was engineered for the first time to produce vitamin A by introducing key enzymes in the β-carotene and retinoid biosynthetic pathways. This approach included integrating genes encoding lycopene cyclase/phytoene synthase (<i>McCarRP</i>), phytoene desaturase (<i>McCarB</i>), and β-carotene 15,15′-dioxygenase (<i>MbBlh</i>). Further optimization of the mevalonate pathway enabled the production of over 600 mg/L vitamin A in a 3-L fed-batch fermentation. Fe²⁺ supplementation improved yield, while a two-phase culture system using dodecane and butylated hydroxytoluene enhanced vitamin A recovery, with over 90% recovered in the extracellular phase.</p> Conclusions <p>This study establishes <i>L. starkeyi</i> as a promising host for sustainable vitamin A production. Although significant improvements in yield were achieved, further optimization of pathway regulation and fermentation conditions is needed to reach economically competitive levels. These findings provide a foundation for developing <i>L. starkeyi</i> as a platform for large-scale production of vitamin A and other valuable terpenoids.</p>