Introduction: Berberine is an important isoquinoline alkaloid that has various pharmacological properties. The comparison of the binding pocket similarity of berberine is regarded as the starting point for deciphering various activities. Eight berberine protein crystals were clustered and studied by molecular dynamics (MD) simulations to investigate common features of berberine binding pockets. Method: Root Mean Square Deviation (RMSD) results showed that berberine was able to bind to each protein in a stable manner. Residue analysis showed that the stability of residue composition of different protein pockets varied. This is also consistent with the results of the pocket similarity analysis: PS-score curves of most proteins fluctuated to varying degrees. The binding pocket of 3BTI in homogeneous protein analysis exhibited high stability (PS-scoremean = 0.703 and PS-scoremin = 0.5664). Pocket similarity analysis between two heterologous proteins showed that most of PS-score values were in the interval of 0.3-0.35, and PS-score values of 3D6Y were relatively high when compared with the other three proteins. Results: Pocket residue matching analysis showed that GLU145/VAL147/ILE182/TYR229/GLU253 in 3D6Y can be matched structurally to the corresponding residues in 1JUM, 2QVD, and 5Y0V, respectively, which can be considered as an important pocket feature for the berberine binding. Nevertheless, the obtained matched residues are limited to the category of pocket structural similarity. Conclusion: This was the first study in which dynamic comparison of berberine binding pockets were used to discover pocket patterns. These results were of great significance for the polypharmacological study, the identification of potential off-targets, and the repurposing of berberine. conclusion: This was the first study in which dynamic comparisons of berberine binding pockets were used to discover interaction patterns. These results were of great significance for the polypharmacological study, the identification of potential off-targets, and the repurposing of berberine.