Plant cell culture has been developed as an alternative method for the production of an anticancer drug, paclitaxel. However, the sensitivity of plant cells to shear stress has been one of the main obstacles to the scale-up of the plant cell culture. To gain a better understanding of the mechanism of plant cells' response to hydrodynamic mechanical stress, lipid profiling of suspension-cultured Taxus chinensis (Chinese yew) var. mairei cells under shear stress was carried out using liquid chromatography-tandem MS and gas chromatography-time-of-flight MS. T. chinensis var. mairei cells cultured in a Couette-type shear reactor responded with an increase of cell membrane permeability compared with control cells, which indicated that the adaptation to shear stress altered membrane lipid composition. The main changes of lipid profiles in the shear-stress-induced cells were the following: (a) the total phospholipid content decreased, especially that of structural phospholipids such as phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine; (b) cells showed increased stigmasterol at the expense of sitosterol and campesterol; (c) the stigmasterol/phospholipid ratio increased; (d) the mono-unsaturated-fatty-acid content increased; (e) the shear-stress-induced cells accumulated very-long-chain saturated fatty acids (C22:0, C23:0, C24:0 and C25:0). These findings suggested alterations in membrane composition and hinted at a possible molecular basis for the mechanotransduction processes induced by shear stress in T. chinensis var. mairei cells.