<p>Selenium (Se) biofortification in forage tree species offers a sustainable strategy to improve nutritional quality. This study investigated the physiological and molecular responses of <i>Broussonetia papyrifera</i>, a high-biomass woody species, to exogenous selenite (Na₂SeO₃) and selenate (Na₂SeO₄). Low Se concentrations (≤ 0.4&#xa0;mM) significantly enhanced plant growth, while higher concentrations (0.8&#xa0;mM), especially Na₂SeO₄, inhibited biomass accumulation in a dose-dependent manner. Both forms of Se substantially increased foliar Se content, with Na₂SeO₄ showing higher uptake efficiency. Genome-wide screening identified four sulfate/Na₂SeO₄ assimilation-related genes including <i>ATP sulfurylase 1</i> (<i>BpAPS1</i>), <i>BpAPS2</i>, <i>adenosine 5′-phosphosulfate reductase</i> (<i>BpAPR1</i>), and <i>BpAPR2</i>. Phylogenetic analyses confirmed the evolutionary conservation of these proteins, which were localized to the chloroplasts. Tissue-specific expression patterns revealed a positive correlation between transcript levels of <i>BpAPS1</i>, <i>BpAPR1</i>, and <i>BpAPR2</i> and Se content, while <i>BpAPS2</i> expression was inversely correlated, indicating functional divergence. Compared to wild-type (WT) plants, transgenic <i>Arabidopsis thaliana</i> lines overexpressing these genes accumulated 1.27- to 1.60-fold more Se under Na₂SeO₄ stress, accompanied by coordinated upregulation of key endogenous Se metabolism genes like <i>AtAPS</i>, <i>AtAPR</i>, <i>S-adenosylmethionine carriers 3</i> (<i>AtSAM</i>), <i>homocysteine S-methyltransferase</i> (<i>AtHMT</i>), <i>methylselenol methyltransferase</i> (<i>AtMMT</i>), suggesting enhanced Se assimilation and detoxification capacity. Collectively, these findings clarify the functional roles of the APS and APR gene families in Se uptake, assimilation, and detoxification in a woody crop, highlighting potential genetic targets for Se biofortification.</p>