Abstract: To investigate the effects of different pretreatment methods on the lipid characteristics and microstructure of Nannochloropsis oceanica, this study employed enzymatic hydrolysis, extrusion puffing, ultrasonic treatment, and microwave treatment for pretreating N. oceanica. The lipid yield, lipid composition, lipid concomitants, antioxidant activity, and microstructural changes of N. oceanica were measured before and after different pretreatments. Results: enzymatic hydrolysis pretreatment group significantly increased lipid yield by 40.46% compared with untreated group and increased total phenolic content by 57.11%; total chlorophyll content reached 1,427.47 mg/100g. However, this pretreatment group also resulted in the highest malondialdehyde (MDA) content (15.95 nmol/mg), suggesting that exposure of unsaturated fatty acids accelerated lipid oxidation. Extrusion puffing pretreatment group resulted in the greatest increase in eicosapentaenoic acid phospholipid content (up to 60.64%) and the highest total tocopherol content (15.80 mg/100 g), accompanied by the most substantial increase in crystallinity. Ultrasonic pretreatment group generated the highest glycolipid content (48.01%). Microwave pretreatment group resulted in lower lipid content compared with enzymatic hydrolysis and extrusion puffing pretreatment group but significantly enhanced carotenoid content (except β-cryptoxanthin), reduced crystallinity, and produced the lowest MDA content (8.51 nmol/mg) among the four pretreatment group, indicating improved oxidative stability. Scanning electron microscopy and Fourier-transform infrared spectroscopy confirmed that all pretreatments effectively disrupted the cell walls of N. oceanica and altered lipid structure. These findings provide a theoretical basis for selecting optimal pretreatment strategies for N. oceanica to tailor lipid properties for food and functional applications.