The application of nitrogen fertilizer within a normal range has been found to increase the leaf nitrogen content and photosynthetic rate of canola plants (Brassica napusL.). Despite numerous studies on the separate effects of CO2diffusion limitation and nitrogen allocation trade-off on photosynthetic rate, few have examined both these factors in relation to the photosynthetic rate of canola. In this study, two genotypes of canola with varying leaf nitrogen content were analyzed to determine the impact of nitrogen supply on leaf photosynthesis, mesophyll conductance, and nitrogen partitioning. The results showed that the CO2assimilation rate (A), mesophyll conductance (gm), and photosynthetic nitrogen content (Npsn) increased with an increase in nitrogen supply in both genotypes. The relationship between nitrogen content andAfollowed a linear-plateau regression, whileAhad linear relationships with both photosynthetic nitrogen content andgm, indicating that the key to enhancingAis increasing the distribution of leaf nitrogen into the photosynthetic apparatus andgm, rather than just increasing nitrogen content. Under high nitrogen treatment, the genotype (QZ) with high nitrogen content had 50.7% more nitrogen than the other genotype (ZY21), but had similarA, which was primarily due to ZY21’s higher photosynthetic nitrogen distribution ratio and stomatal conductance (gsw). On the other hand, QZ showed a higherAthan ZY21 under low nitrogen treatment as QZ had higherNpsnandgmcompared to ZY21. Our results indicate that, in selecting high PNUE rapeseed varieties, it is important to consider the higher photosynthetic nitrogen distribution ratio and CO2diffusion conductance.