Mechanical loading plays an important role not solely in cartilage development, but also in cartilage degeneration. Its adaptation behavior to mechanical loading has not been clearly delineated. The aim of the study was to examine the effect of different running modes (with different muscle contraction types) on morphological, biochemical, and mechanical properties of articular cartilage in the knee of growing rats. Thirty-six female Sprague–Dawley rats were randomly assigned into a nonactive age-matched control (AMC), level (LEVEL), and 20° downhill (DOWN) running group (n = 12 each). Running groups were trained on a treadmill for 30 min/day, 5 days/week for 6 weeks. Immunohistochemical staining and analysis of expression for collagen II, collagen IX, cartilage oligomeric matrix protein (COMP), and matrilin-3, histomorphometry of femoral cartilage height and femoral COMP staining height, and indentation testing of tibial articular cartilage were performed. Rats subjected to downhill running showed a significantly (P = 0.015) higher COMP staining height and a tendentially (P = 0.084) higher cartilage height in the high-weight bearing area of femoral articular cartilage. Cartilage thickness, mechanical properties, and expression of cartilage network proteins in tibial cartilage remained unaffected by different running modes. Our data suggest that joint loading induced by eccentric muscle contractions during downhill running may lead to a site-specific adaptation.