Iron deficiency severely affects crop yield and quality. Gamma-aminobutyric acid (GABA) plays a vital role in plant responses to multifarious stresses. However, the role of GABA in Fe deficiency responses and the potential mechanisms remain largely unknown in cucumber. Here, we found that Fe deficiency raised the GABA levels in leaves and roots of cucumber. To probe the role of GABA in Fe deficiency, the seedlings were subjected to five levels of GABA concentrations (0, 5, 10, 20 and 40 mmol L) for 7 days under Fe deficiency. The results demonstrated that 20 mM GABA in alleviating the Fe deficiency-induced stress was the most effective. GABA pretreatment reduced the Fe deficiency-induced chlorosis and inhibition of photosynthesis and growth, and significantly enhanced the contents of iron in shoots and roots. Exogenous GABA significantly decreased the pH of nutrient solution and increased ferric-chelate reductase (FCR) activity induced by Fe deficiency and the transcript levels of Fe uptake-related genes HA1, FRO2 and IRT1 in roots. GABA also increased the content of auxin (IAA) and expression of auxin biosynthesis (YUC4), response (IAA1), and transport (PIN1) genes under Fe deficiency. Furthermore, exogenous the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) application abolished the GABA-induced changes in Fe deficiency. In summary, we found that GABA improves tolerance to iron deficiency via an auxin-dependent mechanism in cucumber.