It remains unclear whether elevated CO2 would increase N uptake of vegetable and alleviate soil degradation via reducing N residual in greenhouse soils. This study investigated the effect of three CO2 concentrations 400 (ambient CO2), 800 (elevated CO2) and 1200 μmol mol−1 (super-elevated CO2) and two N application rates 0.06 (low N) and 0.24 g N kg−1 (high N) on N uptake of cucumber plants and N cycling in a greenhouse soil in open top chambers. Elevated CO2 increased the biomass and N concentration of fine roots in high N by 24% and 4%, respectively, indicating a greater improvement in N uptake efficiency. The improvement was greater under low N but to a lesser extent under super-elevated CO2. Elevated CO2 altered microbial community as indicated by decreased Shannon diversity, substrate richness and Shannon evenness when averaged N treatments possibly associated with the increased plant N uptake efficiency. Elevated CO2 and super-elevated CO2 tended to decrease gene abundances of soil bacterial amoA, nirS and nosZ in high N with increased plant N uptake and decreased concentrations of NH4+, NO2− and NO3− in soils resulting in less soil N loss. To conclude, moderate CO2 enrichment enhances N uptake efficiency in fine roots of cucumber plants and decreases soil N loss associated with decreased nitrification and denitrification under high N application in a greenhouse soil.