In order to probe the physiological mechanisms of Pyropia haitanensis blade responding to low-salinity stress, in this paper, the next-generation high-throughput sequencing technology was applied to compare transcriptome data of P. haitanensis blades being cultured at normal salinity of 26 (control group) and at low-salinity of 3 (stress group) for 3, 6, and 9 hours, respectively. Results revealed that the de novo assembly of sequence data generated 33 872 unigenes, of which average length was 612 base pairs. Compared with control group, 1 108, 1 638 and 1 881 differentially expressed genes were produced from three stress groups, respectively. The analysis of gene ontology functional enrichment revealed that some important biological processes related to low-salinity stress, such as single-organism metabolic process, monosaccharide biosynthetic process, monosaccharide catabolic process, gluconeogenesis, organic substance catabolic process, etc., got significantly enriched. The results of KEGG pathways enrichment showed that metabolic pathway varied mostly with different treated time. Differentially expressed genes produced from LS 3 h vs LS 0 h group tended to cluster into three KEGG metabolic pathways, one of which related to photosynthesis, and the other two were both interrelated with amino acid biosynthesis. However, under low-salinity stress for 6 and 9 hours, most differentially expressed genes were enriched in metabolic pathways carbohydrate involved. Quantitative real-time PCR validation results showed that expression levels of eight selected differentially expressed genes were consistent with high-throughput sequencing results. All the results above revealed that stress responses of P. haitanensis were time-specific when exposed to low-salinity. In the early stage, P. haitanensis mainly synthesizsed or degraded protein to resist low-salinity environment. With low-salinity stress carried on, cells changed the content of soluble substances and slowed down energy metabolism to resist low-salinity adversity.