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Desert ephemeral plants play an important role in desert ecosystem. Soil water availability is considered as the major restrictive factor limiting the growth of ephemeral plants. Moreover, arbuscular mycorrhizal fungi (AM fungi) are widely reported to improve the growth of desert ephemerals. The present study aimed to test the hypothesis of that AM fungi could alleviate drought stress of desert ephemeral Plantago minuta, and AM fungal functions reduced with the improvement of soil water content. A pot experiment was carried out with three levels of soil water contents (4.5%, 9.0%, and 15.8% (w/w)), and three AM inoculation treatments (Glomus mosseae, Glomus etunicatum and non-inoculation). The results indicate that mycorrhizal colonization rate decreased with the increase of soil water availability. Inoculation improved plant growth and N, P and K acquisition in both shoots and roots regardless water treatments. When comparing the two fungi, plants inoculated with G. mosseae performed better than those inoculated with G. etunicatum in terms of plant growth and nutrient acquisition. These results showed that ameliorative soil water did not suppress arbuscular mycorrhizal fungal functions in improving growth and nutrient acquisition of desert ephemeral Plantago minuta.