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Glyphosate is one of the most widely used herbicides in the world. However, because of its overuse and resistance to degradation, high levels of glyphosate residues in the environment are reported. Therefore, this study aimed to investigate the effects of glyphosate on proteomic aspects of Tetrahymena thermophila and their uses as bioindicators of freshwater ecosystem. First, an acute toxicity test was performed to determine the median inhibition concentration (IC 50 ). The toxicity test results showed that glyphosate inhibited the growth (proliferation) of T. thermophila. The 96 h-IC 50 value of glyphosate was 171 mg L À1 . No visible changes in aggregation behavior and cell morphology were observed under glyphosate exposure. In addition, the effects of low and high dose glyphosate concentrations (77.5 mg L À1 , 171 mg L À1 ) on the proteomic changes of T. thermophila was investigated using a label-free shotgun proteomic approach. A total of 3191 proteins were identified, 2791 proteins were expressed in the control, 2651 proteins were expressed in 77.5 mg L À1 glyphosates, and 3012 proteins were expressed in 171 mg L À1 glyphosates. Under glyphosate exposure at both low and high dose glyphosate, 400 unique proteins were upregulated. The majority of these proteins was classified as proteins associated with oxidative stress response and intracellular transport indicating the shifts in the internal metabolism. Proteomics revealed that the glyphosate metabolism by T. thermophila is a multi-step process involving several enzymes, which can be divided into four phases, including modification (phase I), conjugation (phase II), transport (phase III), and degradation (phase IV). The accumulation of various biochemical reactions contributes to overall glyphosate resistance. With the proteomics approach, we have found that T. thermophila was equipped with glyphosate detoxification and degradation mechanisms.