The impact of the total flavonoids of Citrus aurantium (TFC) treatment on proliferation and apoptosis of 3T3-L1 cells was studied. After 3T3-L1 cells were cultured and treated with TFC in different concentrations for 24 h, the effect of TFC on cell proliferation was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Morphological changes of 3T3-L1 cells were observed under an inverted microscope, the cell apoptotic rate was determined by Annexin V-EGFP/PI double staining, and propidium iodide (PI) staining was used to measure the effect of TFC on cell cycle. The intracellular reactive oxygen species (ROS) level was measured using a ROS assay kit, and the mRNA expression level of apoptosis-related genes was determined by fluorescence quantitative real-time polymerase chain reaction (RT-PCR). The results showed that high concentrations of TFC (300 and 400 μg/mL) could significantly inhibit the proliferation of 3T3-L1 cells; the inhibition rates on cells were 38% and 63%, respectively, and the morphological characteristics of 3T3-L1 cells were changed. The ROS concentration in cells was also increased significantly. The apoptosis experiments indicated that TFC could induce early and late apoptosis of 3T3-L1 cells; the early apoptosis rates for the treatments with 100 μg/mL, 200 μg/mL and 300 μg/mL TFC for 24 h were 4.6%, 15.7% and 22.5%, respectively, and the late apoptosis rates were 14.4%, 8.3% and 32.2%, respectively. The cell cycle results showed that the G0/G1 phase ratio of the treated 3T3-L1 cells decreased from 58.9% to 51.4%%, the ratio of corresponding S phase was increased slightly, and the ratio of the cells in G2/M phase was not significantly changed. The increase in the ratio of pro-apoptotic gene bax and anti-apoptotic gene bcl-2 and the increase in the mRNA expression of apoptosis-related genes p21 and p53 promoted apoptosis in 3T3-L1 cells.