This study highlights the mechanisms involved in Pb(II)-phycoremediation using the Pb(II) tolerant strain of Scenedesmus obliquus. Firstly, monitoring of cell growth kinetics in control and Pb(II)-doped medium revealed a significant growth inhibition, while the analyses through flow cytometry and Zetasizer revealed no difference in cell viability and size. Residual weights of control and Pb(II)-loaded cells assessed by thermogravimetric analysis, were 31.34% and 57.8%, respectively, indicating the uptake of Pb(II) into S. obliquus cells. Next, the use of chemical extraction to distinguish between the intracellular and extracellular uptake indicated the involvement of both biosorption (85.5%) and bioaccumulation (14.5%) mechanisms. Biosorption interaction of the cell wall and Pb(II) was confirmed by SEM, EDX, FTIR, zeta potential, zero-charge pH, and contact angle analyses. Besides, the biochemical characterization of control and Pb(II)-loaded cells revealed that the bioaccumulation of Pb(II) induces significant increases in the carotenoids and lipids content and decreases in the proportions of chlorophyll, carbohydrates, and proteins. Finally, the metabolomic analysis indicated an increase in the relative abundance of fatty acid methyl esters, alkanes, aromatic compounds, and sterols. However, the alkenes and monounsaturated fatty acids decreased. Such metabolic adjustment may represent an adaptive strategy, that preventing high Pb(II)-bioaccumulation in cellular compartments.