DNA J HOMOLOG 3 (J3) is a special transcriptional regulator in flowering time control, but the molecular mechanism of J3 in regulating flowering time has not been thoroughly revealed in B. juncea which is one important oilseed and vegetable crop. In this study, J3 gene was cloned from B. juncea (BjuJ3). Phylogenetic relationship analysis showed that the BjuJ3 had high amino acid sequence similarity (>93%) with other Brassica plants. The BjuJ3-transgenic tobacco plants exhibited early flowering, suggesting that BjuJ3 was an activator of flowering time. The qRT-PCR analysis found that BjuJ3 could be ubiquitously induced by the long-day and vernalization treatments in all the tissues of B. juncea. Yeast two-hybrid assays and GST pull-down experiments revealed that BjuJ3 could not directly interact with BjuSOC1, BjuSVP and BjuAGL24. Whereas, yeast one-hybrid and Dual-Glo (R) Luciferase assays found that BjuJ3 could not interact with BjuAGL24 promoter but could specifically bind to BjuSOC1-1 which is one of truncated fragments of BjuSOC1 promoter. Our research will provide valuable information for unraveling regulatory mechanisms of flowering time in B. juncea. (C) 2018 Elsevier Inc. All rights reserved.