Aquatic environmental pollutants have various impacts on aquaculture. Specifically, sulfide has been established as being toxic to aquatic animals including the oriental river prawn Macrobrachium nipponense. In response, the hepatopancreas has been broadly studied, as it plays a pivotal role in arthropod nutrient digestion and absorption, energy supply, and organ development as well as in crustacean immunity. However, the underlying molecular mechanisms of hepatopancreas’s response to sulfide toxicity are still poorly understand. Herein, we used Nova-seq 6000 platform to conduct a comparative transcriptome analysis of gene expression profiles in the hepatopancreas of M. nipponense, while it was under the influence of a semi-lethal sulfide concentration (3.20 mg/L at 48 h). A total of 139 million raw reads were obtained, in which 67,602 transcripts were clustered into 37,041 unigenes for further analysis. After constant sulfide exposure for 48 h, 235 differentially expressed genes, i.e., DEGs (151 up-regulated and 84 down-regulated) were identified in the sulfide treatment group (TGHP) compared with the control group (CGHP). We used GO and KEGG databases to annotate all the DEGs into 1180 functions and 123 pathways, respectively. The metabolic pathways included proximal tubule bicarbonate reclamation, sulfur metabolism, glycolysis and gluconeogenesis, and the TCA cycle; while immune-related pathways contained Ras, Rap1, focal adhesion and platelet activation. Additionally, apoptosis-involved pathways e.g., lysosome, also exhibited remarkable alteration in the presence of sulfide stress. Notably, responses to external stimuli and detoxification genes— such as GSKIP, CRT2, APOD, TRET1, CYP4C3 and HR39— were significantly altered by the sulfide stress, indicating that significant toxicity was transferred through energy metabolism, growth, osmoregulatory processes and immunity. Finally, we demonstrated that in the present of sulfide stress, M. nipponense altered the expression of detoxification- and extracellular stimulation-related genes, and displayed positive resistance via tight junction activation and lysosome pathways. The results of these novel experiments shed light on the hepatopancreas’s molecular response to sulfide stress resistance and the corresponding adaptation mechanism; and enable us to identify several potential biomarkers for further studies.