The application of nitrogen (N) is a major challenge for sugarcane production due to the low nitrogen use efficiency (NUE). A possible key to increasing the NUE in sugarcane is the adequate supply of molybdenum (Mo) due to their role in nitrate reductase (NR) activity and N metabolism within plant tissue. We hypothesized that Mo supply increases NUE in sugarcane by improving NR activity and dry matter production. Our aim here was to test the combined application of N and Mo on sugarcane plantlets to evaluate the NR activity, biomass production, contents of N and Mo in leaves, and NUE. A growth-chamber experiment was run using N rates (Control; 30; 60; 120 and 240 mg kg−1) and Mo supply (Control and 0.5 mg kg−1) with four replications. Measurements of biomass production, NR activity, N and Mo content in leaves, SPAD index, and NUE were evaluated after 54 days of cultivation. Nitrogen rates increased biomass production, N-content in leaves, SPAD index and NR activity in the absence of Mo supply. Mo supply didn’t affect NR activity in leaves, but increased 48% the NUE at the lowest N rate (30 mg kg−1), suggesting a positive effect of Mo in another pathway of N metabolism. Our results suggest that the Mo application increases NUE under limited conditions of N supply by affecting other N-metabolism process rather than NR activity. Mo uptake increased with N doses, reaching maximum Mo-leaf contents of 17.5 mg kg−1 at the 151.2 mg kg−1 N rate. HIGHLIGHTS Nitrogen rates consistently increases biomass production and N nutrition of sugarcane plantlets Mo supply increases Mo-leaf content but does not affect NR activity Mo supply increases nitrogen use-efficiency under limited N supply.