Few studies have investigated nitrogen (N) fertilizer management in no-tillage (NT) tobacco (Nicotiana tobacum L.) production systems, even though N fertilization is known to influence tobacco cured leaf yield and quality. The present study evaluated how tillage practice and N fertilizer rate affected burley tobacco agronomic performance, plant available nitrogen (PAN) supply, and leaf chemical constituents. In 2012 and 2013, three N fertilizer rates (0, 140 and 280 kg N/ha) were introduced as split-plots within a long-term NT and conventional tillage (CT) (mouldboard plough) comparison study. Results (2007–2013) showed that the effect of tillage on tobacco yield depended on seasonal weather; NT tobacco appeared to have lower yield than CT tobacco in seasons with <450 mm growing season rainfall, but similar yields when rainfall was >500 mm. In 2012 (432 mm rainfall; 84% of the long-term seasonal mean), leaf SPAD reading, leaf nitrate concentration, total nitrogen concentration at the topping day (i.e. removal of flowers/buds at the tops of the plants) and cured leaf nicotine and alkaloid content suggested that N deficiency was more pronounced in NT than CT at the lowest N fertilizer rate. The PAN supply, as measured by a modified in situ resin core method, was similar in 2012 between NT and CT, suggesting that plant factors may have had a role in N uptake efficiency. This scenario did not repeat in 2013 (706 mm rainfall; 137% of the long-term seasonal mean). Even though N fertilization rates were identical for both tillage practices in 2012 and 2013, PAN was lower, on average, in 2012. Because N uptake is largely the result of mass flow, the impact of reduced root density in NT tobacco would be expected to be more pronounced in a season such as 2012, when water was limited. Banding N close to the tobacco root system and/or side-dressing some portion of N may be recommended strategies to improve N use efficiency in NT burley tobacco production.