A xyloglucan-specific endo-1,4β-glucanase (XcXGHA) from Xanthomonas citri pv. mangiferaeindicae has been cloned, expressed in Escherichia coli, purified and characterised. The XcXGHA enzyme belongs to CAZy family GH74 and has catalytic site residues conserved with other xyloglucanases in this family. At its optimal reaction conditions, pH 7.0 and 40 °C, the enzyme has a k cat/K M value of 2.2 × 10(7) min(-1) M(-1) on a tamarind seed xyloglucan substrate. XcXGHA is relatively stable within a broad pH range (pH 4-9) and up to 50 °C (t 1/2, 50 °C of 74 min). XcXGHA is proven to be xyloglucan-specific, and a glycan microarray study verifies that XcXGHA catalyses cleavage of xyloglucan extracted from both monocot and dicot plant species. The enzyme catalyses hydrolysis of tamarind xyloglucan in a unique way by cleaving XXXG into XX and XG (X is xylosyl-substituted glucose; G is unsubstituted glucose), is able to degrade more complex xyloglucans and notably is able to cleave near more substituted xyloglucan motifs such as L [i.e. α-L-Fucp-(1 → 2)-β-D-Galp-(1 → 2)-α-D-Xylp-(1 → 6)-β-D-Glcp]. LC-MS/MS analysis of product profiles of tamarind xyloglucan which had been catalytically degraded by XcXGHA revealed that XcXGHA has specificity for X in subsite -1. The 3D model suggests that XcXGHA consists of two seven-bladed β-propeller domains with the catalytic center formed by the interface of these two domains, which is conserved in xyloglucanases in the GH74 family. However, the XcXGHA has two amino acids (D264 and R472) that differ from the conserved residues of other GH74 xyloglucanases. These two amino acids were predicted to be located on the opposite side of the active site pocket, facing each other and forming a closing surface above the active site pocket. These two amino acids may contribute to the unique substrate specificity of the XcXGHA enzyme.