The pufferfishes Sphoeroides testudineus and Sphoeroides greeleyi are estuarine species that osmoregulate efficiently, but S. testudineus tolerates seawater dilution to a much higher degree than S. greeleyi. This study aimed at testing whether NKCC is involved with their differential tolerance of seawater dilution, through the analysis of in vivo furosemide (NKCC inhibitor) injection both on hypo-regulation (in 35 per thousand salinity) and hyper-regulation (in 5 per thousand salinity). After exposure for 6 h or 5 days to both salinities, blood samples were obtained for determination of plasma osmolality, chloride, sodium and hematocrit, and muscle samples for determination of water content. Furosemide injection led to increased plasma osmolality and sodium in 35 per thousand and decreased osmolality and chloride in 5 per thousand, when compared to saline-injected controls. Furosemide injection led to hematocrit reduction in both salinities, and muscle water content increase in 5 per thousand and decrease in 35 per thousand in S. testudineus. The results are compatible with NKCC working in branchial NaCl secretion in 35 per thousand, in both species, and a higher role in cell volume regulation in blood and muscle cells of S. testudineus, in both salinities, which could partially explain the stronger capacity of S. testudineus to tolerate seawater dilution during low tide.