In order to study the osmoregularatory function of kidney in Scatophagus argus, primary renal cells were treated with hyposmotic, isosmotic and hyperosmotic media for 24 h. The label-free quantitative method was employed in proteomic analysis, and a total of 3,787 proteins were obtained by mass spectrometry. Compared with isotonic group, 14 proteins were significantly expressed in the hypotonic group and 31 in the hyperosmotic group, respectively (Ratio > +/-2 and P < 0.05). Differential expression profiles were analyzed by Gene Ontology (GO) annotations, enrichment analysis and clustering analysis. The proteins associated with cytoskeleton, energy synthesis and protein binding were selected for quantitative detection at mRNA level to confirm changes of renal cells at gene level and protein level. In hyposmotic group, cytoskeleton-related proteins were up-expressed significantly, including tubulin alpha-4A chain, tubulin beta chain, myosin-11 and type I cytoskeletal 18-like. In the hyperosmotic group, significantly expressed proteins were of extracellular matrix, such as fibronectin and collagen. Therefore, it is assumed that after hypo-osmotic stimulation, intracellular osmotic pressure was higher than extracellular osmotic pressure, and the cells expanded immediately, then expressed cytoskeleton related proteins in large quantities, thus affecting other ion channels. Under hyper-osmotic stimulation, the cells were in a state of water loss and morphologic contraction, which affected the adhesion between cells. However, the increase of fn and plod2 showed that the cells were repairing the damaged protein. This study showed changes of protein expression after anisotonic shocks of primary renal cells in Scatophagus argus and may provide references for further studies of osmoregulation.