Nonylphenol (NP) is a biodegradation product of nonylphenol ethoxylates (NPEs) belonging to the alkylphenol ethoxylates (APEs) group. APEs are widely used nonionic surfactants in detergents, herbicides, pesticides, paints, and cosmetics. The present work investigates the effects of NP on rainbow trout (Oncorhynchus mykiss) livers at the molecular level using Fourier transform infrared (FT-IR) spectroscopy. The FT-IR spectra revealed dramatic differences between the NP-treated and control tissues, which mainly indicated that the level of triglycerides increased, the lipid order increased, and the protein concentration decreased in the treated samples. Moreover, it was also found that glycogen levels significantly decreased and the relative content of nucleic acids increased in NP-treated fish. The 17beta-Estradiol-treated fish liver spectra were found to be quite similar to those of NP-treated fish. All these results implied that rainbow trout may offer considerable promise to be used as a bioindicator for NP in the future.