Environmental factors affect photochemical phenotypic plasticity in plants and this is well explained by the Carbon/Nutrient Balance (CNB) hypothesis. CNB describes that carbon based secondary metabolites (CBSCs) are increased under nutrient limitation when growth is reduce and photosynthesis is elevated. Many forest tree species are dependent on symbiotic interaction with ectomycorrhizal fungi for the acquisition of phosphorus (P), which is available in limited amounts in forest soils. To investigate interactions between P deficiency and ectomycorrhizal associations with (Populus tremuloides) seedlings on CBSCs accumulation and photosynthetic capacity, seedlings were grown in greenhouses with or without mycorrhizal fungi (Laccaria bicolor and Paxillus involutus) and with an adequate or a limiting supply of phosphorus for 46 days. Assimilation versus internal CO₂ partial pressure (Ci) curves were used to estimate maximum Rubisco activity (Vc,max) and electron transport mediated ribulose 1,5-bisphosphate regeneration capacity (Jmax). Phosphorous limitation significantly decreased net photosynthetic rate, transpiration, apparent quantum yield, maximum efficiency of PS II (F_v/F_m) and increased nonphotochemical quenching coefficient. Nonmycorrhizal seedlings grown with limiting phosphorus had significantly reduced Vc,max and Jmax compared to seedlings in other treatments.  Mycorrhizal treatment increased photosynthetic capacity in seedlings in the low phosphorus treatment, whereas ectomycorrhizae did not affect the photosynthetic capacity of seedlings in the high phosphorus treatment as compared to nonmycorrhizal seedlings. We conclude that phosphorus supply affects photosynthetic capacity during ectomycorrhizal colonization through effects on Rubisco activity and ribulose 1,5-bisphosphate regeneration rates. Plant biomass under P limitation was significantly reduced in non-mycorrhizal plants as compared to mycorrhizal plants under P limitation. Colonization by L. bicolor and P. involutus and P availability changed CBSC concentrations. These results indicate that the ectomycorrhizal association has specific roles in altering host carbon assimilation and is affected by P supply. Understanding the response of secondary metabolites in plants during symbiotic interactions and under P limitation may provide insight into the missing carbon sink in terrestrial ecosystem.