The overall objective of this study was to determine whether growing season, water supply, and their interaction influence glucosinolate (GSL) concentration and composition in turnip roots (Brassica rapa ssp. rapifera L.). Field experiments on a loamy soil in Großbeeren, Germany, were conducted in the spring‐summer (SS), summer‐autumn (SA), and autumn‐winter (AW) growing seasons. Each experiment included three water‐supply treatments with 25%, 50%, and 75% of available soil water (ASW) as lower thresholds. We found that the total GSL concentration in turnip roots was 1774–3221 μmol (kg fresh matter [FM])–1 and the dominant GSL was aromatic gluconasturtiin (GST) with concentrations of 1004–1628 μmol (kg FM)–1 in turnip roots. Total, aliphatic, and some specific individual GSLs in turnip roots were significantly influenced by water supply, growing season, and their interaction, due to the variations of the root sulfur (S) concentration, climatic conditions, or both. The influence of water supply on GSL concentration was modified by growing season, which in turn influenced S concentration in turnips. In the SS season, the 25%‐ASW water treatment enhanced concentrations of total GSLs by 52% and 47%, aliphatic GSLs by 60% and 131%, and aromatic GSLs by 47% and 21% when compared to the 50%‐ and 75%‐ASW water treatments, respectively. No reduction of root yield was observed, although the shoot yield was reduced by limited water supply. In SA and AW, total GSL concentration did not change under different water‐supply levels, but concentration of individual aliphatic and indole GSLs did. Based on these results, growers can adjust their irrigation and S‐fertilization practices to growing season in order to optimize turnip quality in terms of GSL concentration and composition, while still obtaining higher root yield and enabling better resource utilization.