Microcin J25 (MccJ25) is an antimicrobial peptide produced by a fecal strain of Escherichia coli containing 21 AA. This study was performed primarily to evaluate the effects of MccJ25 as a potential substitute for antibiotics (AB) on growth performance， nutrient digestibility， fecal microbiota， and intestinal barrier function in weaned pigs. In the present study， 180 weaned pigs (7.98 ± 0.29 kg initial BW) were randomly assigned to 1 of 5 treatments， including a basal diet (CON) and CON supplemented with AB (20 mg/kg colistin sulfate; ABD) or 0.5， 1.0， and 2.0 mg/kg MccJ25. On d 0 to 14， dietary supplementation with MccJ25 and ABD had positive effects on ADG， ADFI， diarrhea incidence， and G:F ( < 0.05). Pigs fed the 2.0 mg/kg MccJ25 diet had greater ADG ( < 0.05) and marginally greater G:F ( < 0.10) compared with pigs fed the ABD diet. Compared with the CON diet， the 2.0 mg/kg MccJ25 diet sharply improved ( < 0.05) ADG and G:F and decreased ( < 0.05) diarrhea incidence (d 15 to 28 and d 0 to 28). Apparent digestibility of nutrients in pigs fed 1.0 and 2.0 mg/kg MccJ25 was improved ( < 0.05) compared with that of pigs fed CON and ABD. The serum cytokines IL-6 and IL-1β and tumor necrosis factor-α levels in pigs fed MccJ25 were greater than in pigs fed CON ( < 0.05). Additionally， the IL-10 concentration in pigs fed MccJ25 was sharply increased ( < 0.05) compared with that of pigs fed CON. Pigs fed 1.0 and 2.0 mg/kg MccJ25 diets had remarkably decreased lactate， diamine oxidase， and endotoxin concentrations and fecal numbers ( < 0.05) and improved fecal and numbers ( < 0.05). Compared with the ABD diet， the diet containing 2.0 mg/kg MccJ25 did not increase lactate， diamine oxidase， and endotoxin (d 14) concentrations ( < 0.05) or decrease the and (d 28) numbers ( < 0.05). The diets containing 1.0 and 2.0 mg/kg MccJ25 and ABD (d 28) improved lactate concentration and short-chain fatty acid concentrations， including acetate， propionate， and butyrate， in feces ( < 0.05). Moreover， the pigs fed 2.0 mg/kg MccJ25 had greater lactate， butyrate (d 14)， and propionate concentrations than the pigs fed the ABD diet ( < 0.05). In conclusion， dietary supplemented MccJ25 effectively improved performance， attenuated diarrhea and systematic inflammation， enhanced intestinal barrier function， and improved fecal microbiota composition of weaned pigs. Therefore， MccJ25 could be a potential effective alternative to AB for weaned pigs.