Herein we report the discovery and structure activity relationship (SAR) of a novel metabotropic glutamate receptor 3 (mGlu3) negative allosteric modulators (NAM) probe (ML289) with 15-fold selectivity versus mGlu2 (IC50 >10 μM). The mGlu3 NAM was discovered via a ‘molecular switch’ from a closely related, potent (EC50 = 197 nM) mGlu5 positive allosteric modulator (PAM), CID 16000100. This mGlu3 NAM (CID 56587994, ML289) displays an IC50 value of 649 nM and is inactive on mGlu5 (>30 μM) and clean in a Ricerca ancillary pharmacology panel. ML289 possesses favorable physiochemical properties, a good dystrophia myotonica protein kinase (DMPK) profile and is centrally penetrant. Thus, ML289 is a best-in-class in vitro and in vivo probe for studying non-competitive antagonism of mGlu3.