Abstract: Hydroxyectoine, a remarkable compatible solute renowned for its effective regulation intra- and extracellular osmotic pressure, undergoes stringent regulation in Halophilic bacteria due to its status as an ectoine derivative. Halomonas campaniensis XH26 produces hydroxyectoine when cultured in a medium containing 3% NaCl. This study aims to elucidate the pathways and key factors influencing hydroxyectoine synthesis in H. campaniensis XH26. Utilizing a control group with 0 NaCl and experimental groups with NaCl gradients of 12%, 14% and 16%, we analyzed differentially expressed genes and metabolites across four varying NaCl salinity culture conditions. These analyses were conducted through transcriptomic sequencing and metabolomic profiling using Liquid Chromatograph-Mass Spectrometer (LC-MS). Furthermore, differential gene expression was confirmed through relative quantitative analysis via qPCR. The results showed that the amount of 5-HE synthesis increased and then decreased with the salt gradient and was the highest at 14% NaCl. Transcriptomic analysis identified 186 KEGG metabolic pathways, notably enriched in ABC transporter proteins, the two-component system, and flagellar assembly pathways. Seven key genes implicated in hydroxyectoine biosynthesis-lysC, ectA, ectB, ectC, ectD, doeC, and doeD-exhibited differential expression. Comparative analysis demonstrated that the qPCR validation results were largely consistent with the transcriptomics findings. LC-MS analysis led to the identification of 1159 differential metabolites, among which the compatible solutes ectoine, hydroxyectoine, and betaine showed significant variations. Notably, differences in metabolites were observed in amino acid metabolism, cofactor-vitamin metabolism, and carbohydrate metabolic pathways. In this study, we analyzed the salt tolerance characteristics and salt adaptation mechanism of H. campaniensis XH26. These results preliminarily clarified the patterns of 5-HE production and synthetic gene cluster expression with salinity. In addition, they also provide a theoretical basis for the optimization of wild bacteria with high 5-HE production and the construction of genetically engineered bacteria.