Procambarus clarkii is an economically important species in China; however, its high mortality rate due to pathogenic bacteria, particularly Vibrio parahaemolyticus, results in significant economic loss. This study aimed to understand the immune response of crayfish to bacterial infection by comparing and analyzing transcriptome data of hepatopancreatic tissue from P. clarkii challenged with V. parahaemolyticus or treated with PBS. Physiological indices (TP, Alb, ACP, and AKP) were analyzed, and tissue sections were prepared. After assembling and annotating the data, 18,756 unigenes were identified. A comparison of the expression levels of these unigenes between the control and V. parahaemolyticus groups revealed 4037 DEGs, with 2278 unigenes upregulated and 1759 downregulated in the V. parahaemolyticus group. GO (Gene Ontology) enrichment analysis shows that the DGEs are mainly enriched in cellular anatomical activity, bindinga and cellular process, enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways showed that DGEs were mainly enriched in Base excision repair, Phagosome and Longevity regulating pathway. At the same time, lysosome was also enriched. The phagosome and lysosome pathways play a crucial role in the immune defense of crayfish against V. parahaemolyticus injection that will be highlighted. In addition, the expression levels of six selected immune-related DEGs were measured using qRT-PCR, which validated the results of RNA-seq analysis. This study provides a new perspective on the immune system and defense mechanisms of P. clarkii and a valuable foundation for further investigation of the molecular immune mechanisms of this species.