We present the functional characterization of GlbB, a lysine 4-hydroxylase from the glidobactin biosynthetic gene cluster. Despite its narrow substrate specificity, GlbB is able to catalyze the hydroxylation of l-lysine with excellent total turnover number and complete regio- and diastereoselectivity. The synthetic utility of GlbB is illustrated by its use in the efficient preparation of a key dipeptide fragment of glidobactin.