Comparative proteomics was performed to identify proteins in the cardiac muscle of Takifugu rubripes in initial response to excessive fluoride. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in softwater alone (F(-) = 0.4 mg/L), and the experimental group was raised in the same water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish cardiac muscle and then were subjected to 2D PAGE analysis. The matrix assisted laser desorption ionization time of flight mass spectrometry (Maldi TOF MS) was applied to identify the proteins that were differentially expressed from the two groups of fish. Average of 318 and 275 proteins detected in the control and the experimental group, respectively, 167 spots were matched and 21 highly differentially expressed proteins were further analyzed by Maldi TOF-TOF MS, and 15 were identified by MASCOT. These 15 proteins include Telomerase reverse transcriptase, 4SNc-Tudor domain protein, protein disulfide isomerase ER-60, Tuba1 protein, mitogen-activated protein kinase 10, and SMC4 protein. Consistent with their previously known functions, these identified proteins seem to be involved in apoptosis and other functions associated with fluorosis. These results will contribute to our understanding for the effects of fluoride exposure on the physiological and biochemical functions of takifugu, and the toxicological mechanism of fluoride causing fluorosis in both fish and human.