In an attempt to find a suitable alternative to the otherwise perilous chemical control strategy of disease management, the amino acid proline was evaluated for its efficiency to elicit resistance in pearl millet (Pennisetum glaucum (L.) R. Br.) against downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroet both under greenhouse and field conditions. Proline treatment to seeds at 50 mM concentration for 3 h, significantly enhanced the seed germination and seedling vigor of pearl millet in comparison with the control. The same concentration and duration of seed treatment protected the pearl millet plants from downy mildew by offering 58% protection under greenhouse and 67% protection under field conditions. Studies revealed that 3 days were required for proline-treated plants to develop resistance, which was systemic and was sustained throughout the life of the plants. Apart from disease protection, proline was also found effective in enhancing vegetative and reproductive growth of the plants, as evidenced by the increase in height, fresh weight, leaf area, tillering capacity, 1000-seed weight and grain yield in comparison with the control plants.