Due to its rapid growth rate and excellent adaptability to saline environments, the red tilapia () could be a suitable subject for studies on saline tolerance. In this study, to provide a theoretical basis for saline tolerance mechanisms, we aimed to screen and identify differentially expressed proteins in the gill of Red tilapia using protein chips and mass spectrometry, which were subsequently verified by using immunohistochemistry and western blot techniques. There were 181 differentially expressed protein spots detected (change multiple ≥ 1.5), which included 142 up-regulated proteins and 39 down-regulated proteins. Among these, three proteins were identified by mass spectrometry from five differentially expressed proteins:intermediate filament protein (IF), translocation protein 63 (SEC63), and disulfide-isomerase A3 (PDIA3). The immunohistochemistry results showed that IF and PDIA3 are expressed in the gill base of red tilapia under both freshwater and saline water conditions, and showed a trend of initial decrease and then increase with an increase of saline concentration. However, no positive reaction was detected for SEC63. The western blot results showed IF and PDIA3 expression level changes in different saline waters similar to those observed using immunohistochemistry. In the 30 g/L salinity group, the expression of IF protein decreased during the early stage of stress, reached the lowest value at 48 h, and thereafter began to rise significantly at 72 h, The expression of PDIA3 protein was significantly increased after 96 h ( < 0.05). These results suggest that IF and PDIA3 are positively responsive proteins in the gill tissues of red tilapia under salinity stress, and play important roles in the regulation of cytoskeleton and endoplasmic reticulum function.