This study investigated the impacts of polyether sulfone (PES) substrate with four different molecular weight cut-offs (MWCO) on the performance of the thin-film composite (TFC) nanofiltration membranes. When the MWCO of the substrate increased: (i) the effective thickness of the substrate decreased; (ii) the average pore size of the substrate enlarged in general; and (iii) the surface roughness of the substrate increased. When the MWCO of substrates was increased, the TFC membranes synthesized from them showed the following characteristics: (i) the thickness of the polyamide layer expanded; (ii) average size of the nodules on the membrane surface enlarged, (iii) permeate flux enhanced in general, (iv) rejection of NaCl (RNaCl) increased and rejection of MgSO4 (RMgSO4) did not change significantly; (v) flux recovery rates decreased. The study showed that the MWCO of the substrate had a similar influence on the permeate fluxes produced by the substrate and the TFC membrane. Lower MWCO substrates induced higher selectivity to divalent and monovalent ions.