In the present study, osmoadaptive mechanism of Halomonas sp. AAD12 was studied through analysis of changes in its proteome maps and osmolyte accumulation strategy to understand how this euryhaline microorganism masters osmotic stress of saline environments. Under salt stress, there were significant variations in the expression of proteins involved in osmoregulation, stress response, energy generation and transport. This was accompanied by an increase in proline and hydroxyectoine but a decrease in ectoine accumulation. The major osmolyte at high salinity was proline. Unexpectedly the size of the total ectoines' pool was smaller at elevated salinity. Experimental findings were then integrated with a metabolic model to get insight into carbon trafficking during osmoadaptation. Simulations predicted that the total flux through energy generating pathways, namely gluconeogenesis and the pentose phosphate pathway, was significantly lower and carbon source that entered the system as citrate was mainly diverted to osmolyte synthesis at high salinity. Overall these results suggested that the moderately halophilic Halomonas sp. AAD12 pursued a different osmoregulatory strategy than the two well known moderate halophiles, Chromohalobacter salexigens and Halobacillus halophilus. The climbing value of osmolytes such as ectoine in health care and skin care products places significant attention to halophilic microorganisms hence an understanding of the osmoadaptive mechanism and osmolyte accumulation strategy of this isolate is very valuable to be able to manipulate its metabolism towards desired goals. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.