Root pruning hinders the absorption and utilization of nutrients and water by seedlings in the short term. Arbuscular mycorrhizal fungi (AMF) are an important source of nutrient and water for seedlings except for the root system. However, the mechanism by which AMF affect the physiological growth of seedlings after root pruning has rarely been studied. In this study, a pot experiment was conducted through a three-compartment partition system to clarify the effects of Funneliformis mosseae (F. mosseae) strain BGC XJ07A on the physiological growth of root-pruned Robinia pseudoacacia (R. pseudoacacia) seedlings. Five root pruning treatments (0, 1/5, 1/4, 1/3, and 1/2 of the taproot length was removed) were applied to noninoculated seedlings and those inoculated with F. mosseae. The results showed that the presence of F. mosseae significantly increased the shoot and root biomasses, leaf photosynthetic rate, stomatal conductance, and transpiration rate. The root projected area, root surface area, average root diameter, root density, root volume, and number of root tips of the inoculated seedlings were higher than those without inoculation in all root pruning treatments. The root cytokinin (CTK), gibberellins (GA) and indole-3-acetic acid (IAA) concentrations but root abscisic acid (ABA) concentration were higher than those measured in the absence of inoculation in all root pruning treatments. Moreover, the changes in the root endogenous hormone concentrations of the seedlings were closely related to the root morphological development and seedling biomass. AMF increased the soil available nitrogen, soil available phosphorus, soil available potassium and soil organic matter concentrations compared with the noninoculated treatment. These results indicate that AMF can alleviate the adverse effects of root pruning on the physiological growth of R. pseudoacacia and soil properties and provide a basis for AMF application to forest cultivation and the sustainable development of forest ecosystems.