The GABA transporter 3 (GAT3) is a member of the GABA transporter (GAT) family proposed to have a role in regulating tonic inhibition. The GAT3-preferring substrate (S)-isoserine has shown beneficial effects in a mouse model of stroke accompanied by an increased GAT3 expression, indicating a molecular mechanism mediated by GAT3. However, (S)-isoserine is not ideally suited for in vivo studies due to a lack of selectivity and brain permeability. To elucidate the structural determinants of (S)-isoserine for GAT3 inhibition, and to optimize and inform further ligand development, we here present the design, synthesis and pharmacological evaluation of a series of conformationally constrained isoserine analogs with defined stereochemistry. Using [³H]GABA uptake assays at recombinant human GAT3, we identified the azetidine and pyrrolidine analogs (2S,2´S)-6 and (2S,2´S)-7 as the most potent inhibitors. To further elaborate on the selectivity profile both compounds were tested at all GATs, the taurine transporter (TauT) and GABA_A receptors. Although (2S,2´S)-6 and (2S,2´S)-7 are comparable to (S)-isoserine with respect to potency, the selectivity vs. the taurine transporter was significantly improved (at least 6 and 53 times more activity at hGAT3, respectively). A subsequent comprehensive structure-activity study showed that different connectivity approaches, stereochemical variations, simple or larger α- and N-substituents, and even minor size enlargement of the heterocyclic ring all abrogated GAT3 inhibition, indicating very strict stereochemical and size requirements. The observed structure activity relationships may guide future ligand optimization and the novel ligands ((2S,2´S)-6 and (2S,2´S)-7) can serve as valuable tools to validate the proposed GAT3-mediated effect of (S)-isoserine such as in functional recovery after stroke and thus help corroborate the relevance of targeting GAT3 and tonic inhibition in relevant brain pathologies. [Figure not available: see fulltext.].