<p>Replanting disease caused stunted grapevines growth, hindering the healthy and sustainable development of the grape industry. Nitrogen metabolism is closely related to plant growth, however the effect of replanting on nitrogen metabolism in grape have seldom been reported. In this study, we used ‘Beta’ grape (<i>Vitis riparia</i> × <i>V. labrusca</i> cv.) cuttings as experimental materials to investigate the nitrogen content, nitrogen uptake behavior, and ammonium transporter genes (<i>AMT</i>) expression levels in grapevines after replanting. The results indicated the total nitrogen content in both the roots and leaves of the replanted grapevines decreased. After replanting, the net influx rate of NO₃⁻ into grapevine roots decreased, whereas that of NH₄⁺ increased. The Ndff (Nitrogen derived from fertilizer) of ¹⁵N-NH₄⁺ in the roots of replanted grapevines increased, and the ¹⁵N-NH₄⁺ distribution ratio in the stems of replanted grapevines decreased compared to that in the CK. In total, 12 <i>AMT</i> genes were identified in the grape genome. The RT-qPCR results revealed that the transcript levels of the 12 <i>VlAMT</i> genes in grapevine roots displayed dynamic changes, and <i>VlAMT2.1</i> and <i>VlAMT4.1</i> increased significantly on the 96th d after replanting. Further studies showed that <i>VlAMT4.1</i> is localized on the plasma membrane and has ammonium transport function. Therefore, grapevines altered the expression level of the <i>AMT</i> gene in roots under replanting conditions and exhibited a preference for the uptake of ammonium nitrogen.</p>