In this study, a 1H nuclear magnetic resonance (NMR) based metabolomics approach was applied to investigate the toxicity of acetamiprid) in Gambusia holbrooki (Mosquitofish). Acetamiprid is commonly used both in agriculture and domestic areas against a wide range of insects that have gained resistance to other pesticides such as  organophosphate, carbamate and pyrethroid.The commercial formulation of acetamiprid (RastT 20 SP) supplied from a local agricultural pest store, were used in this study. Static renewal acute toxicity tests were performed in glass dishes containing 5 L test solution with 10 fish/vessel. Initially, the 96-h LC50value of acetamiprid was estimated for G. holbrooki.  Sublethal concentrations of tested pesticide were applied (1/2, 1/5 and 1/10of 96-hLC50value) for 96-h. The 24-h, 48-h, 72-h, 96-h LC50 value were determined for Acetamiprid was as 75.9, 54.1, 48.5, 42.2 mg active ingredient/L respectively. 1H NMR spectra of acetamiprid is recorded for its solution in CDCl3 and D2O. The obtained signals suggest the signals thermodynamically not stable. The NMR spectra in CDCl3 could be taken more easily because of the higher solubility in this solvent. Another advantage of this solvent was the absence of signal overlapping around 3.4, 3.5 and 3.8 ppm The signals in D2O are more complex because of signal overlapping around 3.1 ppm.