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Lipids are the essential components of the cell intracellular and plasma membranes. Sulfoquinovosyldiacylglycerol (SQDG) is a glycolipid; glycolipids can replace phospholipids in maintaining phosphate (Pi) homeostasis in plants which are undergoing Pi starvation. Sulfoquinovosyl diacylglycerol synthase 1 (OsSQD1) is a critical enzyme in the first step of catalyzation in the formation of SQDG in rice. In this study, the expression pattern of different zones in roots of <i>OsSQD1</i> in response to different Pi conditions is examined, and it is found that <i>OsSQD1</i> is highly expressed in lateral roots under Pi-sufficient and -deficient conditions. The root phenotype observation of different <i>OsSQD1</i> transgenic lines suggests that the knockout/down of <i>OsSQD1</i> inhibits the formation and growth of lateral roots under different Pi conditions. Additionally, the lipid concentrations in <i>OsSQD1</i> transgenic line roots indicate that <i>OsSQD1</i> knockout/down decreases the concentration of phospholipids and glycolipids in Pi-starved roots. The <i>OsSQD1</i> mutation also changes the composition of different lipid species with different acyl chain lengths, mainly under Pi-deprived conditions. The relative transcript expression of genes relating to glycolipid synthesis and phospholipid degradation is estimated to help study the mechanism by which <i>OsSQD1</i> exerts an influence on the alteration of lipid composition and concentration in Pi-starved roots. Moreover, in Pi-starved roots, the knockout of <i>OsSQD1</i> decreases the unsaturated fatty acid content of phospholipids and glycolipids. To summarize, the present study demonstrates that <i>OsSQD1</i> plays a key role in the maintenance of phospholipid and glycolipid composition in Pi-deprived rice roots, which may influence root growth and development under Pi-deprived conditions.