Since the advent of Huanglongbing [HLB (Candidatus Liberibacter asiaticus)] in Florida, several preliminary reports have emerged about the positive effects of mineral nutrition on the performance of HLB-affected citrus (Citrus sp.) trees. HLB-affected trees are known to undergo significant feeder root loss. Therefore, studies have focused on foliar nutrient application instead of soil-applied nutrients speculating that the HLB-affected trees root systems may not be competent in nutrient uptake. Some studies also suggest that HLB-affected trees benefit from micronutrients at higher than the recommended rates; however, the results are often inconclusive and inconsistent. To address this, the goal of the present study was to evaluate the nutrient uptake efficiency and the quantitative and qualitative differences in nutrient uptake of HLB-affected trees. HLB-affected and healthy sweet orange (Citrus sinensis) trees were grown in a 100% hydroponic system with Hoagland solution for 8 weeks. The trees were deprived of any fertilization for 6 months before the transfer of trees to the hydroponic solution. Altogether, the four treatments studied in the hydroponic system were healthy trees fertilized (HLY-F) and not fertilized (HLY-NF), and HLB-affected trees fertilized (HLB-F) and not fertilized (HLB-NF). HLY-F and HLY-NF trees were found to have similar levels of leaf nutrients except for N, which was found to be low in nonfertilized trees (HLY and HLB). Both HLB-F and HLB-NF trees had lower levels of Ca, Mg, and S compared with HLY trees. In addition, HLB-NF trees had significantly lower levels of micronutrients Mn, Zn, and Fe, compared with HLY-NF trees. The hydroponic solution analysis showed that HLB-F and HLY-F trees had similar uptake of all the nutrients. Considering that HLB-affected trees have a lower root-to-shoot ratio than healthy trees, nutrient uptake efficiency per kilogram of root tissue was significantly higher in HLB trees compared with HLY trees. Under nutrient-deficient conditions (day 0) only nine genes were differentially expressed in HLB roots compared with HLY roots. On the other hand, when fertilizer was supplied for ≈1 week, ≈2300 genes were differentially expressed in HLB-F roots compared with HLY-F roots. A large number of differentially expressed genes in HLB-F were related to ion transport, root growth and development, anatomic changes, cell death, and apoptosis compared with HLY-F trees. Overall, anatomic and transcriptomic analyses revealed that HLB-affected roots undergo remarkable changes on transitioning from no nutrients to a nutrient solution, possibly facilitating a high uptake of nutrients. Our results suggest the roots of HLB-affected trees are highly efficient in nutrient uptake; however, a small root mass is a major limitation in nutrient uptake. Certain micronutrients and secondary macronutrients are also metabolized (possibly involved in tree defense or oxidative stress response) at a higher rate in HLB-affected trees than healthy trees. Therefore, a constant supply of fertilizer at a slightly higher rate than what is recommended for micronutrients and secondary macronutrients would be beneficial for managing HLB-affected trees.