The orientation of a β-sheet membrane peptide in lipid bilayers is determined, for the first time, using two-dimensional (2D) 15N solid-state NMR. Retrocyclin-2 is a disulfide-stabilized cyclic β-hairpin peptide with antibacterial and antiviral activities. We used 2D separated local field spectroscopy correlating 15N-1H dipolar coupling with 15N chemical shift to determine the orientation of multiply 15N-labeled retrocyclin-2 in uniaxially aligned phosphocholine bilayers. Calculated 2D spectra exhibit characteristic resonance patterns that are sensitive to both the tilt of the β-strand axis and the rotation of the β-sheet plane from the bilayer normal and that yield resonance assignment without the need for singly labeled samples. Retrocyclin-2 adopts a transmembrane orientation in dilauroylphosphatidylcholine bilayers, with the strand axis tilted at 20°±10° from the bilayer normal, but changes to a more in-plane orientation in thicker 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC) bilayers with a tilt angle of 65°±15°. These indicate that hydrophobic mismatch regulates the peptide orientation. The 2D spectra are sensitive not only to the peptide orientation but also to its backbone (ϕ, ψ) angles. Neither a bent hairpin conformation, which is populated in solution, nor an ideal β-hairpin with uniform (ϕ, ψ) angles and coplanar strands, agrees with the experimental spectrum. Thus, membrane binding orders the retrocyclin conformation by reducing the β-sheet curvature but does not make it ideal. 31P NMR spectra of lipid bilayers with different compositions indicate that retrocyclin-2 selectively disrupts the orientational order of anionic membranes while leaving zwitteronic membranes intact. These structural results provide insights into the mechanism of action of this β-hairpin antimicrobial peptide.