Milkfish (Chanos chanos) is an aquaculture species which could be cultivated in the rearing ponds with different salinities. In the Southern Taiwan, large numbers of milkfish died in the cold current of the winter leading to economic loss of the fisherman. Being an ectothermal species, low temperature seriously influences the homeostasis of the milkfish. In the teleosts, the gills exposed to the external environment are the major osmoregulatory organ. Mitochondrion-rich (MR) cells conduct ion excretion and uptake by ion transporters in gills of seawater (SW) and fresh water (FW) fish, respectively. The present study focused on the remodeling of cellular morphologies and modulation of ion transporters in the gill MR cells of SW and FW milkfish raised in the non-lethal cold stress (18°C) for 1 week compared to the control temperature (28°C) group. The juvenile milkfish (15-20 g, n=12) were fed a daily diet of commercial pellets. Following the experiment, the fish were not fed for 1 day and anaesthetized with Tricaine mesylate (MS-222, 100-200 mg/l) before collecting blood and excising the gill tissues. The facilities and protocols for experimental animals were approved by the Institutional Animal Care and Utility Committee of the National Chung-Hsing University (Approval No. IACUC 96-48 to THL). Values were mean ± SEM, compare by Students’ t-test. The plasma osmolality (28°C vs. 18°C: 403.4±4.2 vs. 377.0±3.1 mOsmo/kg, p<0.05) and Na+ concentrations (28°C vs. 18°C: 141.1±3.6 vs. 128.3±3.4 mM, p<0.05) were decreased in SW milkfish exposed to 18°C, while the Cl- concentrations (28°C vs. 18°C: 108.6±6.4 vs. 175.6±5.9 mM, p<0.05) were increased in the FW fish at 18°C. No effect of nonlethal cold was found on the protein abundance of Na+, K+-ATPase (NKA) α-subunit and cell size and density of NKA-immunoreactive (IR)/MR cells in gills of the milkfish. In gills of the SW milkfish exposed to 18°C, constant NKA activity and the low percentage of cystic fibrosis transmembrane conductance (CFTR)-IR/NKA-IR cells (28°C vs. 18°C: 43.8±5.5 vs. 14.6±1.6 %, p<0.05) were found. On the other hand, our results revealed that the NKA activity decreased (28°C vs. 18°C: 36.8±6.4 vs. 18.7±4.2 μmol Pi/mg protein/hr, p<0.05) and the apical openings with Na+, Cl- cotransporter (NCC) of MR cells reduced (28°C vs. 18°C: 18.5±1.6 vs. 9.7±1.3 %, p<0.05) in gills of the FW milkfish at 18°C. This study was the first to figure out different responses of MR cells with hypo- and hyperosmoregulation in gills of the milkfish when exposed to nonlethal cold stress. The functional morphologies of gill MR cells played an important role in cold resistance of the euryhaline milkfish.