This study aimed to test whether ectomycorrhizal fungi and exogenous Ca²⁺ have collaboratively influence on the growth trait of Pinus sylvestris var. mongolica seedlings. During our study, several research methods like mycology, biochemistry, and ecology were used. And the appropriate concentration of exogenous Ca²⁺ was applied to one-year-old mycorrhizal seedlings, then the growth and physiological traits of various parts of seedlings were tested. The results indicated that:(1)Appropriate concentration of exogenous Ca²⁺ associated with ECMF could better promote the growth of P. sylvestris var. mongolica seedlings. When Ca concentration ≤ 20mmol/L, the plants height reached to the maximum, which were 11.4cm and 10.6cmseparately,andwere 32.6% and 23.3% higher than control, which treated without applying Ca concentration. Both of biomass and photosynthetic pigment contents were higher than the treatments without applying Ca concentration. The results above showed that an appropriate concentration of exogenous Ca could magnify the effect of mycorrhizal on growth of seedlings. (2) Appropriate concentration of exogenous Ca²⁺ associated with ECMF could better promote the antioxidant ability of P. sylvestris var. mongolica seedlings. By applying exogenous Ca²⁺, the CAT and POD activities in every part of seedlings were increased, and the quality of seedlings were improved due to the high levels of antioxidant enzyme system. (3) Appropriate concentration of exogenous Ca²⁺ associated with ECMF could better promote the resistance to penetration of P. sylvestris var. mongolica seedlings. In the paper, by using exogenous Ca²⁺ together with ECMF could effectively increase the content of soluble protein of P. sylvestris var. mongolica seedlings. The PCA analysis indicated that, the soluble protein have the greatest contribution among all osmolytes indicators. The result also showed that exogenous Ca²⁺ and ECMF could lead to the increasement of content of soluble protein in plants, and their stress resistance was improved as well. (4) It is possible to achieve the goal of promoting the growth and physiological traits of seedlings by applying the appropriate concentration of exogenous Ca²⁺. In conclusion, apparently, the variation trends of growth and physiological traits were similar, the internal antioxidant enzyme system and osmolytes content were higher in the rapid growth and strong seedlings. In the early seedling stage, by applying the appropriate concentration of exogenous Ca²⁺ (or calcium fertilizer) associated with ECMF in the greenhouse or field would obtain better strong seedling results, and this method is feasible.