Identified and validated BnaA03.XTH4 as a negative regulator of plant height through QTL mapping and RNA-seq, with promoter structural variation influencing expression, providing a foundation for dwarf breeding in B. napus. Rapeseed (Brassica napus L.) is an important oilseed crop that faces yield limitations due to lodging susceptibility. Plant height (PH) significantly affects seed yield and lodging resistance; however, the genetic mechanisms remain largely elusive. In this study, we developed multiple genetic populations derived from a dwarf line DZ and a tall line GW. Through simple sequence repeat mapping and specific-locus amplified fragment sequencing, we identified twenty-three preliminary quantitative trait loci, explaining 0.82%-27.22% of the phenotypic variation. Integrated analysis pinpointed four major intervals, PHA02, PHA03, PHA09, and PHC04. RNA sequencing analysis revealed differential expression of BnaA03.XTH4, encoding a xyloglucan endotransglucosylase/hydrolase, in DZ versus GW lines. Promoter analysis uncovered a structural variation associated with enhanced transcriptional activity of BnaA03.XTH4 in dwarf lines. Subcellular localization confirmed BnaA03.XTH4 localizes to the cell wall and apoplast. Heterologous overexpression in Arabidopsis thaliana demonstrated a dual function of BnaA03.XTH4, as a negative regulator of PH and stress response. These findings offer new insights into the regulatory mechanisms of PH and offer molecular targets for improving rapeseed breeding strategies.