In the process of cultivating drought-tolerant plants by using molecular breeding technology, it is necessary to understand the drought resistance mechanism of plants and possess a large number of candidate genes. Drought-tolerant plants are excellent materials for understanding drought tolerance mechanisms and mining drought-tolerant genes. In this study, Ammopiptanthus nanus was used as the material. Polyethylene glycol (PEG-6000) was used to simulate drought stress. The photosynthetic pigments content, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), soluble sugar, soluble protein, and proline (Pro) content in the leaves of A. nanus seedlings were analyzed at 0, 1, 3, 6, and 12 h after PEG treatment. Meanwhile, the gene expression changes of A. nanus under the drought were analyzed by using RNA-Seq technology. The results indicated that the contents of soluble sugar, Pro, and photosynthetic pigments in the leaves of A. nanus were increased under drought stress. SOD and POD responded quickly to drought, and their activities were increased significantly, which played an important role in eliminating excessive reactive oxygen species caused by drought. However, CAT did not respond effectively to drought within 12 hours. Transcriptome analysis identified 15188 differentially expressed unigenes, among which there were 805 co-differentially expressed unigenes. The number of up-regulated unigenes was similar to that of down-regulated unigenes. In the Gene Ontology (GO) enrichment analysis, these 805 unigenes were highly enriched in pathways such as ribulose-1,5-bisphosphate carboxylase/oxygenase and negative regulation of abscisic acid-activated signaling pathway. The four pathways with the smallest P-value (chlorophyll binding, photosystem II, photosynthesis, and light harvesting, photosystem I) are all related to photosynthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the co-differentially expressed unigenes are mainly enriched in six metabolic pathways including photosynthesis - antenna proteins and carbon fixation in photosynthetic organisms. In A. nanus leaves, 7-hydroxymethyl chlorophyll a reductase was continuously down-regulated under drought stress, which inhibiting the degradation of chlorophyll and effectively protecting the photosynthetic system. This study also focused on the signal transduction pathways of plant hormones and finds that the abscisic acid signaling pathway had changed significantly. In addition, in A. nanus leaves, the response of jasmonate ZIM-domain protein (JAZ) to drought was significantly different from that of other species. This study provides new insight into the adaptation mechanisms of A. nanus under drought environment from physiological and molecular level and lays a foundation for the subsequent cloning and functional verification of the drought-related candidate genes.