Microgels have emerged as promising building blocks for constructing porous membranes due to their soft, porous, responsive, and functional nature. In this work, we describe the fabrication of a versatile zwitterionic microgel-based membrane, comprising poly(N-isopropylacrylamide-co-N-(Acryloyloxy)succinimide) (poly(NIPAm-co-NAS)) microgels covalently cross-linked with zwitterionic N,N,N-tris(2-aminoethyl)-3sulfo-1-propanaminium salt (TRIPS) through a nucleophilic reaction between the succinimidyl ester group and the primary amine group. The poly(NIPAm-co-NAS) (PNN-X) microgels, with varying compositions of NIPAm and NAS monomers, were synthesized via a surfactant-assisted precipitation copolymerization. Subsequently, membranes were constructed by vacuum filtration of a dilute microgel suspension over a track-etched support followed by cross-linking with TRIPS. After thorough structural, morphological, and physicochemical characterizations, filtration and rejection properties were assessed. The nanostructured microgel membranes exhibited excellent water permeation with smart gating properties, owing to the high-water absorptivity and thermoresponsiveness of the microgel. The nanosized pores, formed due to the densely packed microgels and its own cross-linked structure, enabled strong rejection of low molecular weight dyes and macromolecules. Furthermore, the pore size tunability under external stimuli provided the capability for stepwise separation of different solutes with varying diameters by controlling their molecular transmission through the membrane. In summary, the prepared nanoporous zwitterionic microgel membrane exhibits strong antifouling properties with smart permeation and molecular separation, signifying its potential for water purification and various environmental separation and filtration.