The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram‐negative bacterium in an ATP‐dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase‐like domain at its N‐terminus. C39 domains are thiol‐dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39‐like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39‐like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB.