Soil Zn is always potentially deficient in Yangtze River Basin (YRB) of China, due to widespread alkaline soils, high soil P, and large Zn removal of high-yielding oilseed rape (Brassica napus L.) and rice (Oryza sativa L.) cultivars. However, Zn fertilizer response of winter oilseed rape in YRB is not clear. Therefore, on-farm experiments were conducted at six locations to investigate the effects of experimental sites and Zn rates (0 and 6.8 kg Zn ha(-1)) on plant growth, seed yield and quality, and Zn uptake of oilseed rape (Huashuang no. 5 cultivar). Across all the sites, Zn application increased seed yield by 5.0 to 16.0%, oil yield by 6.2 to 19.5%, and protein yield by 2.3 to 17.7%. Application of Zn fertilizer increased the concentrations of total oil, oleic acid, and linoleic acid in rapeseed, but reduced stearic acid concentration. No differences were observed on protein and linolenic acid concentrations between Zn rates. Total Zn uptake in plants increased by 13.5 to 62.3% with Zn fertilizer applied, while Zn harvest index reduced by 3.4 to 18.5%. Oilseed rape performance varied among sites. The extensive significant Zn x site interactions indicated that Zn response was dependent on the environments we studied. Thus, Zn fertilizer management will probably need to be adjusted at different sites. Our results suggested to apply Zn fertilizer to improve oilseed rape productivity, nutritional quality, and Zn uptake. This study also increased the understanding of the positive effects of Zn on rapeseed oil content and fatty acids composition.