Euglena gracilis is a unique microalga that lacks a cell wall and is able to grow under different trophic culture conditions. In this study, cell growth, biomass production, and changes in the ultrastructure of E. gracilis cells cultivated photoautotrophically, mixotrophically, and under sequential-heterotrophy-photoinduction (SHP) were assessed. Mixotrophy induced the highest cell growth and biomass productivity (6.27 ± 0.59 mg/L/d) in E. gracilis, while the highest content of fatty acids, 2.69 ± 0.04% of dry cell weight (DCW) and amino acids, 38.16 ± 0.08% of DCW was obtained under SHP condition. E. gracilis also accumulated significantly higher saturated fatty acids and lower unsaturated fatty acids when cultivated under SHP condition. Transcriptomic analysis showed that the expression of photosynthetic genes (PsbA, PsbC, F-type ATPase alpha and beta) was lower, carbohydrate and protein synthetic genes (glnA, alg14 and fba) were expressed higher in SHP-culture cells when compared to other groups. Different trophic conditions also induced changes in the cell ultrastructure, where paramylon and starch granules were more abundant in SHP-cultured cells. The findings generated in this study illustrated that aerobic SHP cultivation of E. gracilis possesses great potential in human and animal feed applications.