As a pivotal regulatory molecule, long non-coding RNA (lncRNA) can coordinate the network regulation of transcription and post-transcriptional processes. However, little is known about their cis-regulatory contributions in gene expression of crustaceans under alkalinity stress. In this study, the purpose was to explore the potential role of lncRNA in gill of Pacific white shrimp (Litopenaeus vannamei) under different alkalinity stress (C: 50 mg/L and T: 350 mg/L). We found 5034 high confidence lncRNAs in gills. Among them, 82 differentially expressed lncRNA (DELs) in C and T groups may affect gill stress by regulating gene expression in cis- or trans- mode at transcriptional level. The cis-functions DELs were enriched in several pathways, including Glutathione metabolism, Peroxisome and Lysosome. The trans-functions DELs were enriched in Toll-like receptor signaling pathway, Peroxisome and Lysosome. We also obtained 12 DELs-mRNA pairs consisting of 6 DELs and 11 mRNAs, and DELs have potential for positively affecting their neighboring genes, predominantly in a cis-regulatory manner. In addition, we found that two hub genes, ARSB and gba-3, were significantly enriched in Lysosome. In the silencing experiment, XR_003476973.1 and MSTRG.20723.1 were found to regulate the Lysosomal pathway under alkalinity stress through cis-acting. Under alkalinity stress, physiological parameters related to homeostasis regulation, immune response and lysosomal hydrolysis changed significantly. These results provide new insights into the mechanism of lncRNA in shrimp gills under alkaline stress, and also offer valuable perspectives into the implications of gill regulation for crustaceans in saline-alkaline water.