β-ketoacyl-ACP synthase II (KASII)is a key enzyme responsible for the conversion of palmitic acid (16∶0-ACP)to stearic acid (18∶0-ACP). KASII activity determines the level of C18 fatty acids. In this paper, Dunaliella salina was used to isolate and identify the DsKASII gene. Bioinformatics tools were used to analyze the subcellular localization, protein structure, physicochemical properties and phylogenetic characteristics of DsKASII enzyme. Detail examinations were conducted for DsKASII gene expression pattern, oil/fatty acid profiles, the contents of chlorophyll and β-carotene under nitrogen deficiency stress. The results showed that Dunaliella salina DsKASII enzyme protein had a length of 476 aa and a theoretical isoelectric point of 6.99. The protein contained plastid-targeting peptide and many hydrophilic regions. The main secondary structures included α-helix (22.48%), β-sheet (22.06%), and random coil (55.46%). The 3D structure simulation predicted that the DsKASII was compactly heart-shaped structure on the whole, with a homodimer as the functional form. Phylogenetic analysis indicated that DsKASII protein has the highest homology with Chlamydomonas reinhardtii CrKASII by 99%. It is possible that the two microalgae have a common evolutionary ancestor. Quantitative RT-PCR (qRC-PCR)analysis revealed that the expression level of DsKASII gene was up-regulated in algal cells under nitrogen deficiency stress compared with the normal culture controls. The expression level of DsKASII in the stressed cells was 4.5 times higher than that in the normal culture cells on the 3th day of cultivation. Moreover, under nitrogen stress, the total oil, oleic acid (C18∶1)and carotenoid content in algae cells increased significantly, whereas palmitic acid (C16∶0)and chlorophyll contents decreased. Taken together, the data indicated that nitrogen stress induced the up-regulation of DsKASII gene in Dunaliella salina, and then DsKASII catalyzed more palmitic acid into stearic acid, eventually increasing the accumulation of monounsaturated oleic acid and carotenoids. The present study provides a scientific reference for the further analysis on the mechanism underlying accumulation of oil and carotenoids as well as algal response to the stress, benefiting the breeding of high-quality oleaginous microalgae.