Procambarus clarkii is an important aquatic organism in China, but its aquaculture has suffered great economic losses due to pathogen infections. To better understand the P. clarkii immune response, we used RNA sequencing (RNA-seq) to examine the expression responses of its hemocyte transcriptome to lipopolysaccharide (LPS). Through assembly and annotation, a total of 53,910 unigenes were identified, with an average length of 1246 bp. 589 differentially in total expressed genes (DEGs) were obtained by the injection of LPS with 310 upregulated genes and 279 downregulated genes. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified several immune response pathways. Additionally, a lot of DEGs to do with the Ras signaling pathway, lysosome, Rap1 signaling pathway, and mitogen-activated protein kinase signaling pathway were upregulated after LPS challenge. Results of Real-time quantitative reverse transcription PCR revealed that 11 randomly selected immune response genes were upregulated after LPS stimulation compared to phosphate-buffered saline stimulation, and this result also validated the RNA-seq data. Our data further enrich the transcriptome databases of P. clarkii and provide a basis for further analysis of the immune system and defense mechanisms of P. clarkii against LPS challenge. Unlabelled Image • The hemocytes transcriptomes of Procambarus clarkii were constructed using high-throughput sequencing. • Totally 53,910 unigenes was obtained from the transcriptome. • Totally 589 DGEs were identified including 310 up- and 279 down-regulated unigenes. • The qRT-PCR results of the transcriptome validation candidate gene are consistent with the transcriptome. [ABSTRACT FROM AUTHOR]