Photosynthetic-nitrogen use efficiency (PNUE) is a useful trait to characterize leaf economics, physiology, and strategy. In this study, we investigated the differences in PNUE, leaf nitrogen (N) allocation, and mesophyll conductance (gm) in Dalbergia odorifera and Erythrophleum fordii (N-fixing trees), and Betula alnoides and Castanopsis hystrix (non-N-fixing trees). Seedlings of the four species were cultured in pots and received the same nutrient solution, water volume, and light. LiCor-6400 was used to determine fluorescence yield, photosynthetic response to light, and intercellular CO2 concentration (Ci). N allocation fractions in the photosynthetic apparatus were calculated according to Niinemets and Tenhunen method; gm was calculated according to variable J, EDO, and A-Ci curve fitting methods. PNUE of D. odorifera and E. fordii were significantly lower than those of B. alnoides and C. hystrix because of their allocation of a lower fraction of leaf N to Rubisco (PR) and bioenergetics (PB). Mesophyll conductance had a significant positive correlation with PNUE in D. odorifera, E. fordii, and B. alnoides. The fraction of leaf N to cell wall (PCW) had a significant negative correlation with PR in B. alnoides and C. hystrix. We conclude that B. alnoides and C. hystrix optimized their leaf N allocation toward photosynthesis, with the trade-off being N allocation to the cell wall and Rubisco. Thus, these two species may have a higher competitive ability in natural ecosystems with fertile soil.