Vesicles with molecular permeability have attracted considerable attention as biomedical materials, e.g., as biocatalytic nanoreactors for drug delivery and artificial cells. However, their applications are limited by their low permeability for enzyme substrates. Here, we report the synthesis of oligo(aspartic acid)-b-poly(propylene oxide) polymer vesicle nanoreactors with a negatively charged surface, which are preferentially permeable for cationic and neutral compounds. The permeation of cationic substrates is accelerated by the electrostatic effect, which increases the apparent rate of the enzymatic reaction. Notably, the polymer can be incorporated into a phospholipid membrane, where it acts as a synthetic molecular channel. The obtained results clearly suggest that imparting the vesicle surface with an anionic charge represents a simple and versatile approach to substrate sorting and enhances molecular permeability. This study can thus be expected to open new avenues for the design of vesicles with molecular permeability that may serve as biocatalytic nanoreactors in artificial cells and drug delivery applications.