The variable region of camelid heavy‐chain antibodies produces the smallest known antibody fragment with antigen‐binding capability (a VHH). The VHH R303 binds internalin B (InlB), a virulence factor expressed by the pathogen Listeria monocytogenes. InlB is critical for initiation of Listeria infection, as it binds a receptor (c‐Met) on epithelial cells, triggering the entry of bacteria into host cells. InlB is surface‐exposed and is required for virulence, hence a VHH targeting InlB has potential applications for pathogen detection or therapeutic intervention. Here, the expression, purification, crystallization and X‐ray diffraction of R303 are reported. Crystals of R303 were obtained following in situ proteolysis with trypsin. Gel filtration and SDS–PAGE revealed that trypsin removed the C‐terminal tag region of R303, facilitating crystal formation. Crystals of R303 diffracted to 1.3 Å resolution and belonged to the monoclinic space group P21, with unit‐cell parameters a = 46.4, b = 31.2, c = 74.8 Å, β = 93.8°. The crystals exhibited a Matthews coefficient of 1.95 Å3 Da−1 with two molecules in the asymmetric unit.