Brassica napus (oilseed rape) is a globally important crop with a wide range of uses such as vegetable oil, animal feed, and alternative fuel. To maximize the field yield, N-fertilizers are often overused in modern agriculture and thus causing nitrate leaching and finally affecting the whole ecosystem. Here we aim to increase the nitrogen use efficiency (NUE) of oilseed rape by modifying its lateral roots structure. To do that, a profound understanding of regulation of lateral roots emergence and development is required. In this study, we first used a chemical N-1-naphthylphthalamic acid (NPA, an auxin transport inhibitor) to inhibit the emergence of lateral roots. And then used another chemical 1-naphthalene acetic acid (NAA, exogenous auxin to break the NPA inhibition) to induce as well as synchronize the lateral roots initiation. For 24 time points within the following 72 hours, time-series RNA-seq data were obtained allowing us to construct a Brassica napus lateral root gene regulatory network (LR-GRN).