Increased availability of monomeric aluminum (Al3+) in forest soils is an important adverse effect of acidic deposition that reduces root growth and inhibits nutrient uptake. There is evidence that Al3+ exposure interferes with NO3− uptake. If true for overstory trees, the reduction in stand demand for NO3− could increase NO3− discharge in stream water. These effects may also differ between species that tolerate different levels of soil acidity. To examine these ideas, we measured changes in relative uptake of NO3− and NH4+ by six tree species in situ under increased soil Al3+ using a 15N-labeling technique, and measured soluble soil Al levels in a separate whole-watershed acidification experiment in the Fernow Experimental Forest (WV). When exposed to added Al3+, the proportion of inorganic N acquired as NO3− dropped 14% across species, but we did not detect a reduction in overall N uptake, nor did tree species differ in this response. In the long-term acidification experiment, we found that soluble soil Al was mostly in the free Al3+ form, and the concentration of Al3+ was ~65 μM higher (~250%) in the mineral soil of the acidified watershed vs. an untreated watershed. Thus, increased levels of soil Al3+ under acidic deposition cause a reduction in uptake of NO3− by mature trees. When our 15N uptake results were applied to the watershed acidification experiment, they suggest that increased Al3+ exposure could reduce tree uptake of NO3− by 7.73 kg N ha−1 year−1, and thus increase watershed NO3− discharge.