Pervaporation technology using PEBA-2533 membranes was investigated to separate a low concentration of pyridine from the ternary mixture of pyridine, water, and NaCl. The experimental results showed that the studied membrane could achieve the enrichment of pyridine and the rejection of NaCl simultaneously. The permeation of pyridine was driven by the temperature and pyridine concentration in the feed solution. The experimental results for the effect of NaCl content on the fluxes correlated with the simulation values of water and pyridine activities. The pyridine activity values increased and water activity decreased on adding NaCl to the feed solution. What is more, it was confirmed that the accumulation of NaCl in the membrane increased with the NaCl concentration, which could influence the flux. According to the results of molecular dynamics (MD) simulations, the introduction of NaCl reduced the number of hydrogen bonds between pyridine and water, which was beneficial to the separation. The long-term operation tests for the pervaporation process showed that the poly(ether-block-amide) (PEBA) membrane had excellent stability, which proved the feasibility of pervaporation as an industrial process.