Low nitrogen stress severely impedes crop growth and productivity. There has been substantial research on root adaptation to low nitrogen conditions in many plant species. However, the mechanism underlying the morphological response of the strawberry (Fragaria × ananassa Duch.) root to low-NO3− or low-NH4+ stress remains poorly understood. Strawberry plants were hydroponically cultivated under 1 mM NO3−, 1 mM NH4+, and control (15 mM NO3−) conditions to assess the physiological responses of their roots to low nitrogen stress. As a result, low nitrogen stresses increased the fresh weight of root, lateral root density, and root surface area, as well as enhanced the accumulation of indole-3-acetic acid and jasmonic acid while significantly reducing salicylic acid in the roots. Correspondingly, low nitrogen stresses increased PM H+-ATPase activity. Low-NO3− stress enhanced the activities of nitrate reductase and glutamine synthetase, whereas low-NH4+ treatment led to higher glutamine synthetase and glutamate synthase activities. Collectively, the present results demonstrate that low nitrogen stresses enhance nitrogen uptake of strawberry roots by regulating hormones (indole-3-acetic acid, jasmonic acid, and salicylic acid) and thereby mediating PM H+-ATPase activity, while promoting nitrogen metabolism by upregulating the activities of nitrate reductase, glutamine synthetase, and glutamate synthase. In conclusion, low nitrogen conditions may facilitate more efficient acquisition of available N from the soil by strawberry root system.