Nitrate nitrogen (NO3−-N) is widely used in the cultivation of the cucumber (Cucumis sativusL.). In fact, in mixed nitrogen forms, partially substituting NO3−-N with NH4+-N can promote the absorption and utilization of nitrogen. However, is this still the case when the cucumber seedling is vulnerable to the suboptimal-temperature stress? It remains unclear as to how the uptake and metabolism of ammonium affect the suboptimal-temperature tolerance in cucumber seedlings. In this study, cucumber seedlings were grown under suboptimal temperatures at five ammonium ratios (0NH4+, 25%NH4+, 50%NH4+, 75%NH4+, 100%NH4+) for 14 days. Firstly, increasing ammonium to 50% promoted the growth and root activity and increased protein and proline contents but decreased MDA content in cucumber seedlings. This indicated that increasing ammonium to 50% enhanced the suboptimal-temperature tolerance of cucumber seedlings. Furthermore, increasing ammonium to 50% up-regulated the expression of the nitrogen uptake-transport genesCsNRT1.3,CsNRT1.5andCsAMT1.1, which promoted the uptake and transport of nitrogen, as well as the up-regulation of the expression of the glutamate cycle genesCsGOGAT-1-2,CsGOGAT-2-1,CsGOGAT-2-2,CsGS-2andCsGS-3, which promoted the metabolism of nitrogen. Meanwhile, increased ammonium up-regulated the expression of the PM H+-ATP genesCSHA2andCSHA3in roots, which maintained nitrogen transport and membranes at a suboptimal temperature. In addition, 13 of 16 genes detected in the study were preferentially expressed in the roots in the increasing ammonium treatments under suboptimal temperatures, which, thus, promoted nitrogen assimilation in roots to the enhance the suboptimal-temperature tolerance of cucumber seedlings.