Agronomic practices, including tillage, crop rotation and N fertilization, have been developed to efficiently manage soil N dynamics and crop N nutrition. These practices can affect soil organic carbon (SOC) and soil total nitrogen (STN) sequestration, and consequently influence soil nitrogen mineralization (SNM) and crop N nutrition. However, little research has been systematically and simultaneously conducted to examine the effect of agronomic management on (1) SOC and STN stocks; (2) SNM; and (3) crop N nutrition. Burley tobacco (Nicotiana tobacum L.) is a N demanding crop and subject to inefficiency in N fertilization. Moreover, conservation tillage and rotation have been integrated into traditionally tillage intensive tobacco cropping systems. Thus, a tobacco tillage and rotation study was used to test how agronomic practices can affect N dynamics and crop N status in a series of sequential experiments. Firstly, different tobacco production systems were utilized to investigate the effects of tillage and rotation on soil aggregate stabilization and associated SOM sequestration. No-tillage and rotation management enhanced SOC and STN stocks, mainly by increasing the proportion of macroaggregates and SOC and STN concentrations. Secondly, a series of studies were conducted on SNM, including: (1) comparison of laboratory and in situ resin-core methods in estimating SNM; (2) evaluation of the influence of N fertilizer application on SNM; and (3) comparison of chemical indices for predicting SNM across management treatments over time. Laboratory method had different results relative to in situ method due to sample pretreatments. Fertilizer N application had a priming effect on SNM, but priming depended on both the N fertilizer rate and the background SOM level. The effect of rotation/tillage treatments on SNM was stable across years and SOC appeared to be the best indicator of SNM among other soil carbon and N estimates. Thirdly, a N fertilizer study for different tillage systems was conducted in 2012 and 2013. Crop parameters and plant available N (PAN) were collected to investigate the impact of tillage on tobacco production. Crop parameters showed that no-tillage can result in N deficiency in dry years. Similar PAN for both tillage methods suggested N deficiency in no-till tobacco was due to the crop’s lower N uptake capacity. In 2014, tobacco root analysis confirmed that no-tillage can result in less root exploration of the soil volume than conventional tillage.