Quantitative magnetic resonance (MR) relaxation parameters demonstrate varying sensitivity to the orientation of the ordered tissues in the magnetic field. In this study, the orientation dependence of multiple relaxation parameters was assessed in cadaveric human cartilage with varying degree of natural degeneration, and compared with biomechanical testing, histological scoring, and quantitative histology. Twelve patellar cartilage samples were imaged at 9.4 T MRI with multiple relaxation parameters, including T 1 , T 2 , CW − T 1ρ , and adiabatic T 1ρ , at three different orientations with respect to the main magnetic field. Anisotropy of the relaxation parameters was quantified, and the results were compared with the reference measurements and between samples of different histological Osteoarthritis Research Society International (OARSI) grades. T 2 and CW − T 1ρ at 400 Hz spin-lock demonstrated the clearest anisotropy patterns. Radial zone anisotropy for T 2 was significantly higher for samples with OARSI grade 2 than for grade 4. The proteoglycan content (measured as optical density) correlated with the radial zone MRI orientation anisotropy for T 2 (r = 0.818) and CW − T 1ρ with 400 Hz spin-lock (r = 0.650). Orientation anisotropy of MRI parameters altered with progressing cartilage degeneration. This is associated with differences in the integrity of the collagen fiber network, but it also seems to be related to the proteoglycan content of the cartilage. Samples with advanced OA had great variation in all biomechanical and histological properties and exhibited more variation in MRI orientation anisotropy than the less degenerated samples. Understanding the background of relaxation anisotropy on a molecular level would help to develop new MRI contrasts and improve the application of previously established quantitative relaxation contrasts.