The ribosome-dependent mRNA interferase YafO from Escherichia coli belongs to a type II toxin-antitoxin (TA) system and its cognate antitoxin YafN neutralizes cell toxicity by forming a stable YafN-YafO complex. The YafN-YafO TA system is upregulated by the SOS response (a global response to DNA damage in which the cell cycle is arrested and mutagenesis is induced) and may then inhibit protein synthesis by endoribonuclease activity of YafO with the 50S ribosome subunit. Structural information on the YafN-YafO complex and related complexes would be helpful in order to understand the structural basis of the mechanism of mRNA recognition and cleavage, and the assembly of these complexes. Here, the YafN-YafO complex was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 3.50 Å resolution and belonged to the hexagonal space group P622, with unit-cell parameters a = 86.14, b = 86.14, c = 173.11 Å, α = β = 90, γ = 120°. Both Matthews coefficient analysis and the self-rotation function suggested the presence of one molecule per asymmetric unit in the crystal, with a solvent content of 65.69% (V(M) = 3.58 Å(3) Da(-1)).