MXene membranes has emerged as promising membrane materials due to their rigid lamellar structure and easily functionalized terminal groups. Understanding the mechanism of solvent transportation through MXene membrane at the microscopic level is significant for the development of new MXene based membranes. In this study, MXene membranes with different interlayer d-spacings (e.g., 0.9 nm, 1.45 nm, 2.0 nm, and 2.5 nm) are investigated for organic solvent nanofiltration (OSN) of four solvents (e.g., acetone, acetonitrile, methanol, and ethanol) and a model solute (Nile red). In membrane with the d-spacing of 0.9 nm, the solvent fluxes are regulated by the arrangements of solvents in the membrane. An ordered orientation and arrangement of solvent would result in a high flux. At this time, the solvent arrangement is dominated by the interaction energies between the solvent and membrane surface. When the d-spacing is larger than 1.45 nm, the solvent property (e.g., viscosity) plays the dominant role in flux determination. Compared with reported membranes, a substantially higher solvent flux can be achieved through the 2D MXene lamellar membranes. The solute rejections are controlled by a size-sieving mechanism, and 100% rejection of Nile red could be obtained in MXene membranes with 0.9 and 1.45 nm d-spacings. From the bottom-up, this work reveals the key governing factors that dominate the solvent permeation and solute rejection of 2D MXene lamellar membranes, and would also promote the development of new OSN membranes.