α-Galactosidases (EC 22.214.171.124) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activitybased probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-D-galactopyranoside substrate (Km = 0.17 mM) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.