The filter paper method was used to determine the diameter of inhibition zone （DIZ） of berberine against Escherichia coli， Bacillus subtilis， Staphylococcus aureus and Salmonella， and the minimum inhibitory concentration （MIC） was determined by the double dilution method to evaluate its antibacterial activity. The stability of berberine was determined by studying the effects of temperature， pH and ultraviolet on the rate of change of berberine's antibacterial activity. In addition， the influence of berberine on bacterial protein was determined by measuring the protein concentration inside and outside the bacterial cell and performing polyacrylamide gel electrophoresis （SDS-PAGE）. The results showed that berberine had certain antibacterial effects on the four tested bacteria. The diameter of the inhibition zone against Escherichia coli， Bacillus subtilis， Staphylococcus aureus and Salmonella was （11.6±0.11）mm， （10.1±0.18）mm， （10.2±0.14） mm and （9.8±0.14） mm respectively. The minimum inhibitory concentration is 2.40， 3.60， 3.30 and 3.945 mg/mL respectively. The antibacterial effect on Escherichia coli is more significant. Ultraviolet almost had no effect on the antibacterial activity of berberine. Strong acid， strong alkali or high temperature can destroy the structure of part berberine and reduced its antibacterial activity； The intracellular protein concentration of bacteria added with berberine decreased， while the extracellular protein concentration increased. Calculation of the total concentration of cell protein and SDS-PAGE analysis showed that berberine can inhibit protein synthesis.