Manila grass (Zoysia matrella), a warm-season turfgrass, usually wilts and browns by late autumn because of low temperature. To elucidate the molecular mechanisms regarding Manila grass responses to cold stress, we performed transcriptome sequencing of leaves exposed to 4°C for 0 (CK), 2h (2h_CT) and 72h (72h_CT) by Illumina technology. Approximately 250 million paired-end reads were obtained and de novo assembled into 82,605 unigenes. A total of 34,879 unigenes were annotated by comparing their sequence to public protein databases. At the 2h- and 72h-cold time points, 324 and 5,851 differentially expressed genes (DEGs) were identified, respectively. Gene ontology (GO) and metabolism pathway (KEGG) enrichment analyses of DEGs indicated that auxin, gibberellins, ethylene and calcium took part in the cold signal transduction in the early period. And in the late cold period, electron transport activities, photosynthetic machinery and activity, carbohydrate and nitrogen metabolism, redox equilibrium and hormone metabolism were disturbed. Low temperature stress triggered high light, drought and oxidative stress. At the physiological level, cold stress induced a decrease in water content, an increase in levels of total soluble sugar, free proline and MDA, and changes in bioactive gibberellins levels, which supported the changes in gene expression. The results provided a large set of sequence data of Manila grass as well as molecular mechanisms of the grass in response to cold stress. This information will be helpful for future study of molecular breeding and turf management.