In cyanobacteria and most Archaeplastida, Arg regulates its formation via allosteric inhibition of the controlling enzyme, N-acetyl-L-glutamate kinase (NAGK) that requires PII protein to properly sense the feedback inhibitor. Although PII expression has been shown to be reduced in Dunaliella salina compared to other green algae, the potential impact of this protein on DsNAGK activity remains unclear. We here performed coupled enzyme assay and surface plasmon resonance analysis and show that DsNAGK is activated by NAG and inhibited by Arg but is not controlled by DsPII. Moreover, DsPII has likely lost its function as an effective glutamine sensor. Replacement of the C-terminus from DsPII with the C-terminus from Chlamydomonas PII restored sensitivity to glutamine in a recombinant DsPII protein, demonstrating the importance of C-terminal residues close to the Q-loop for PII functions. The findings are discussed in the context of the relationship between NAGK control and the acquisition of salinity tolerance during evolution.