Excreted antibiotics into intestine after parenteral administration may be unwanted and can disrupt the indigenous microbiota and cause some adverse clinical consequences. To mitigate this adverse effect, we developed BL, a recombinant beta-lactamase formulated into enteric-coated pellets, to degrade the excreted beta-lactam antibiotics. Herein, we reported its preclinical efficacy evaluation in rats, using ampicillin and cefotiam as model antibiotics. In one study, animals were assigned to receive oral vehicle or different dosage of BL(24∼144μg/dose) three times around one intravenous dose of antibiotics, blood and intestinal samples were collected, and antibiotics concentration were determined. In the second study, animals received daily intraperitoneal antibiotics concurrently with vehicle or different dosage of BL(72∼216μg/dose) 3 times per day , or no treatment, for three consecutive days, fecal samples were collected before and after the antibiotics treatment and cultured for bifidobacterium/lactobacillus counting. Herein we showed that orally administered BL significant decreased the antibiotics intestinal concentration with no noticeable influence to the serum concentration. Antibiotics’ treatment induced a 5-6 order of magnitude decrease in bifidobacterium/lactobacillus count, and BL intervention could prevent gut from such damage and maintain the counts similar to the no treatment group. These preclinical studies demonstrated that BL could degrade the excreted beta-lactam antibiotics and protect against its damage to gut probiotic bacteria.