The C : G base-pair at position -17 in the spacer of β-lactamase gene promoter was removed by restriction with BspH Ⅰ. partial filling-in with Klenow fragment, and trimming with niung bean nuclease. The β-lactamase activities of both bacteria harboring the wildtype and C-17 deleted plasmids were determined using ampicillin as substrate, and the tolerances of the bacteria to ampicillin were tested. The results indicate that the C-17 deletion increases the promoter strength by about 60%. The mutant has more resistance to ampicillin. The half-inhibition concentration of ampicillin for the mutant growth is 280μg/ml. At the same concentration, the wild-type cell density is only about half as much as that of the mutant. The causes for the promoter-up mutation were discussed.