We investigated the change mRNA expression of GtHs subunits (FSHβ, LHβ) in the pituitary, androgen receptor (AR), estrogen receptor (ERα) in gonad and histological observation of gonads in longthooth grouper Epinephelus bruneus by treatment Femara, an aromatase inhibitor (AI). Longtooth grouper (body weight 408±43.1 g; one year) cultured in Future Aquaculture Research Center, NFRDI were used in the experiments. The experiment was conducted for 12 weeks from 21 August 2013. Fish received intramuscular injection of AI at 5 mg/g BW dose in three times every 3 weeks. Fish were sampled pituitary and gonads at 3, 6, 12 weeks post-injection (n=50). The mRNA levels of FSH-β, LH-β in pituitary and AR, ERα mRNA in gonad were evaluated using qRT-PCR and qPCR. The histological change of gonads observed on light microscope. The gonads of control group contained most perinucleolus oocyte. At 3 to 6 weeks post-injection, the gonads of AI-treated group contained a few degenerated oocytes, spermatogonia and spermatocytes. At 12 weeks post-injection, gonads contained spermatids undergoing spermatogenesis. From 6 to 12 weeks post-injection, the expression level of GtHs subunits mRNA in pituitary was significantly higher than control group. The expression level of AR mRNA in gonad was higher than control group from 3 to 12 weeks post-injection. The expression level of ERα mRNA in gonad was lower than control group from 6 to 12 weeks post-injection. These results suggest that immature longtooth grouper with AI treatment induced masculinization via change of GtH subunits in pituitary, AR and ERα mRNA in gonad.