Guppy (Poecilia reticulata) can adapt to a wide range of salinity changes. To investigate the gene expression changes in the guppy exposed to seawater, we characterized its gill transcriptome using RNA sequencing. Experimental fish were exposed to salinity increase from 0 to 30‰ within 4 days, while control fish were cultured in freshwater (0‰ salinity). Seven days after salinity exposure, the gills were sampled and the mortality within 2 weeks was recorded. No significant difference in the cumulative mortality at the second week was found between the two groups. Transcriptomic analysis identified 3477 differentially expressed genes (DEGs), including 1067 upregulated and 2410 downregulated genes. These DEGs were enriched in several biological processes, including ion transport, ion homeostasis, ATP biosynthetic process, metabolic process, and immune system process. Oxidative phosphorylation was the most activated pathway. DEGs involved in the pathway “endoplasmic reticulum (ER)-mediated phagocytosis,” “starch and sucrose metabolism,” and “steroid biosynthesis” were mainly downregulated; chemokines and interleukins involved in “cytokine-cytokine receptor interaction” were differentially expressed. The present results suggested that oxidative phosphorylation had essential roles in osmoregulation in the gills of seawater acclimated guppy, during which the decline in the expression of genes encoding V-ATPases and calreticulin had a negative effect on the phagocytosis and immune response. Besides, several metabolic processes including “starch and sucrose metabolism” and “steroid biosynthesis” were affected. This study elucidates transcriptomic changes in osmotic regulation, metabolism, and immunity in seawater acclimated guppy.