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Boron (B) is essential for plant growth, but toxic in excess. In several countries, soil toxic B levels are always a severe agricultural problem in arid and semi-arid regions. Phytoremediation of excess B containing soil is still in its infancy, while high B tolerant plants with elevated protein abundance of B efflux transporter were successfully established or explored. Brassica napus (B. napus) is one of the most important oil crops. However, B efflux transporters underlying excess B tolerance in B. napus remain unknown. Here, we reported that in Brassicaceae species, B. napus had four homologous genes of Arabidopsis AtBOR4 , which were renamed BnaBOR4.1 , BnaBOR4.2 , BnaBOR4.3 and BnaBOR4.4. BnaBOR4.1 , BnaBOR4.2 and BnaBOR4.3 showed constitutive expression and BnaBOR4.4 appeared to be a pseudogene. BnaBOR4.2 and BnaBOR4.3 were expressed in inner cell layers and BnaBOR4.1 in the outer cell layer in root tip, and all were expressed in vascular tissue in the mature zone. B efflux activity assays in yeast demonstrated that BnaBOR4.1, BnaBOR4.2 and AtBOR4 but not BnaBOR4.3 had comparable levels of B transport activity. Structure-functional analysis between BnaBOR4.3 and BnaBOR4.2 demonstrated that amino acid residue substitution at position 297 (Ala vs Pro) and 427 (Met vs Leu) is critical for the B transport activity. Mutant BnaBOR4.3M427L partially restored the B efflux activity, and both mutants BnaBOR4.3A297P and BnaBOR4.3A297P&M427L fully restored B efflux activity, indicating that the Pro297 residue is critical for their function. Further validation of BnaBOR4 was accomplished by growing transgenic Arabidopsis plants under high B conditions. Taken together, our study identified two functional B efflux genes BnaBOR4.1 and BnaBOR4.2 in B. napus , and a key amino acid residue proline 297 associated with B efflux activity. This study highlights the potential of BanBOR4 genes for B. napus cultivation under high B stress. • Brassica napus Westar 10 showed enhanced excess B (Boron) tolerance than Arabidopsis thaliana Col-0. • BnaBOR4.1 and BnaBOR4.2 were identified as functional B efflux transporters. • Evolutionary variations of amino acid residues at positions 297 and 427 determined B transport activity between BnaBOR4.2 and BnaBOR4.3. • The spatial coordination of BnaBOR4.1 and BnaBOR4.2 ensured efficient B output in Brassica napus roots. [ABSTRACT FROM AUTHOR]