In order to determine an optimal fertilization depth for winter oilseed rape (Brassica napus L.), barrel experiment was conducted in 2013—2014 and 2014—2015 with six treatments, including no fertilizer (F0), and fertilization depths of 0cm (CK), 5cm (D5), 10cm (D10), 15cm (D15) and20cm (D20). The effects of different fertilization depths on above-ground dry weight of taproot and lateral root, and the length, volume, area of lateral root, nutrient uptake and yield of winter oilseed rape were systematically analyzed and compared. The results showed that D15 treatment significantly increased the taproot dry weight in 5~20cm and deeper than 20cm soil depths, while the reduction of taproot dry weight in 0~5cm soil depth was not great compared with CK and D5, thus the total taproot dry weight was significantly increased. The maximum lateral root dry weight, length, volume and area in the 10~20cm soil layer at both seedling stage and flowering stage were found in D15 treatment, which were also significantly higher than those in CK and D5. Above-ground dry matter and shoot nutrient uptake in D15 were the highest at both flowering and harvest stages of all the treatments, and D15 treatment achieved the greatest rapeseed yield and the most N, P, K uptakes in the seed at harvest, while the above-ground dry matter, shoot nutrient uptake, rapeseed yield in CK and D5 were significantly lower than those in D15 treatment. Compared with CK, the N, P, K uptakes of shoot at harvest in D15 treatment across the two growing seasons were increased by 48.07%, 52.18%, 62.96% and 25.75%, 30.19%, 33.41% in comparison with D5 treatment, respectively. In two growing seasons, the yield in D15 treatment was 85.10%, 45.47% and 31.26% higher than those in CK, D5 and D10, respectively. Considering the root distribution, nutrient uptake and yield of winter oilseed rape among all the treatments, fertilization depth of 15cm (D15) could be recommended as the optimal fertilization depth in oilseed rape cultivation.