BACKGROUND: Dark-field radiography imaging exploits the wave character of x-rays to measure small-angle scattering on material interfaces, providing structural information with low radiation exposure. We explored the potential of dark-field imaging of bone microstructure to improve the assessment of bone strength in osteoporosis. METHODS: We prospectively examined 14 osteoporotic/osteopenic and 21 non-osteoporotic/osteopenic human cadaveric vertebrae (L2-L4) with a clinical dark-field radiography system, micro-computed tomography (CT), and spectral CT. Dark-field images were obtained in both vertical and horizontal sample positions. Bone microstructural parameters (trabecular number, Tb.N; trabecular thickness, Tb.Th; bone volume fraction, BV/TV; degree of anisotropy, DA) were measured using standard ex vivo micro-CT, while hydroxyapatite density was measured using spectral CT. Correlations were assessed using Spearman rank correlation coefficients. RESULTS: The measured dark-field signal was lower in osteoporotic/osteopenic vertebrae (vertical position, 0.23 ± 0.05 versus 0.29 ± 0.04, p < 0.001; horizontal position, 0.28 ± 0.06 versus 0.34 ± 0.04, p = 0.003). The dark-field signal from the vertical position correlated significantly with Tb.N (ρ = 0.46, p = 0.005), BV/TV (ρ = 0.45, p = 0.007), DA (ρ = -0.43, p = 0.010), and hydroxyapatite density (ρ = 0.53, p = 0.010). The calculated ratio of vertical/horizontal dark-field signal correlated significantly with Tb.N (ρ = 0.43, p = 0.011), BV/TV (ρ = 0.36, p = 0.032), DA (ρ = -0.51, p = 0.002), and hydroxyapatite density (ρ = 0.42, p = 0.049). CONCLUSION: Dark-field radiography is a feasible modality for drawing conclusions on bone microarchitecture in human cadaveric vertebral bone. RELEVANCE STATEMENT: Gaining knowledge of the microarchitecture of bone contributes crucially to predicting bone strength in osteoporosis. This novel radiographic approach based on dark-field x-rays provides insights into bone microstructure at a lower radiation exposure than that of CT modalities. KEY POINTS: Dark-field radiography can give information on bone microstructure with low radiation exposure. The dark-field signal correlated positively with bone microstructure parameters. Dark-field signal correlated negatively with the degree of anisotropy. Dark-field radiography helps to determine the directionality of trabecular loss.