Glyoxylate reductase (GLYR) is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory glyoxylate and succinic semialdehdye, an intermediate of the γ-aminobutyrate (GABA) pathway. Two isoforms of GLYR exist in plants, GLYR1 and GLYR2, and while GLYR2 is known to be localized in plastids, GLYR1 has been reported to be localized in either peroxisomes or the cytosol. Here, the intracellular localization of Arabidopsis GLYR1 was reappraised by conducting microscopy-based experiments that address some novel mechanisms by which proteins can be directed to peroxisomes. For instance, the C-terminal tripeptide sequence of GLYR1, -SRE, despite its resemblance to a type 1 peroxisomal targeting signal, was not sufficient for peroxisomal targeting. Collectively, the results define the cytosol as the intracellular location of GLYR1 and provide a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol.