Aims The purpose of this study was to isolate Lactobacillus from gastrointestinal tract of healthy postweaning piglets and investigate its synergistic antimicrobial and probiotic effects with ZnO nanoparticles (nZnO). Methods and Results Of the 128 isolates, Lactobacillus plantarum BLPL03 was selected based on its excellent acid and bile salt tolerance properties. Lactobacillus plantarum BLPL03 was sensitive to beta-lactams, macrolides, amphenicols and cephalosporins, whereas it displayed the steady resistance to aminoglycosides, tetracyclines, quinolones and peptide antibiotics. In vitro analysis of antibacterial activities showed that L. plantarum BLPL03 inhibited the four common food-borne pathogenic bacteria including Escherichia coli O157:H7 CMCC 44828, Salmonella Typhimurium ATCC 13311, Staphylococcus aureus CMCC 26003 and Listeria monocytogenes CMCC 54007 in synergy with nZnO. Furthermore, the quantitative polymerase chain reaction test demonstrated that the combined administration of L. plantarum BLPL03 fermentation liquor (LFL) and nZnO synergistically elevated the faecal number of Bifidobacterium by 73 center dot 19-fold, and reduced the two potential enteropathogenic bacteria Enterobacteriaceae and Clostridium perfringens in mice challenged with Salm. Typhimurium. Finally, dietary supplementation with low dose of nZnO (20 mg kg(-1)) when combined with LFL administration enhanced final body weight, fur appearance and average daily gain, and decreased feed conversion ratio and diarrhoea incidence in weaned piglets. The faecal Bifidobacterium and Lactobacillus of piglets were dramatically enhanced by 81 center dot 96- and 3 center dot 15-fold, respectively, after administration of a mixture of nZnO and LFL. Meanwhile, combination of nZnO with LFL resulted in low levels of Bacteroides, Enterococcus, and Enterobacteriaceae. Conclusions A combination of nZnO and LFL exhibits potential health-benefit properties for the control of gut microbial composition by their synergistic antimicrobial and probiotic effects. Significance and Impact of the Study This study may provide a potential nutritional strategy to improve performance and gut health of animals with gut microbiota disorders caused by pathogen infections and weanling, and so on.