Matrine (MT) is a potential resistance reversal agent. However, it remains unclear whether MT can reverse the resistance of ( ) to β-lactams, and, if so, by what mechanism MT works. We screened one cefaclor (CEC)-resistant strain (clinical strain C7) from eight clinical ( ) strains and determined the underlying resistance mechanism. Then, we investigated the reversal effect of MTon the resistance of this strain to CEC. The production of β-lactamase, overexpression of AcrAB-TolC system, and formation of biofilm might not be responsible for the resistance of clinical strain C7 to CEC. Fourteen mutation sites were found in four PBP genes ( , and ) of clinical strain C7, among which the mutation sites located in (Y D and L R) and (A V) genes triggered the resistance to CEC. The minimum inhibitory concentration (MIC) of CEC against clinical strain C7 was reduced by two to eight folds after MT treatment, accompanied by the significant down-regulated expression of mutated and genes. Based on such results, we believed that MT could reverse the resistance of to CEC by inhibiting the mutations in and genes. Our research would provide useful information for restoring the antimicrobial activity of β-lactams and improving the therapeutic efficacy of Glässer's disease.