MicroRNAs (miRNAs) have important roles in many cellular processes, including cell proliferation, growth and development and disease control. Previous study demonstrated that the expression of two highly homologous miRNAs (miR-192 and 215) was upregulated in weaned piglets with Escherichia coli F18 infection. However, the potential molecular mechanism of miR-192 in regulating E. coli infection remains unclear in pigs. In this study, we analyzed the relationship between level of miR-192 and degree of E. coli resistance using transcription activator-like effector nuclease (TALEN), in vitro bacterial adhesion assays and target genes research. A TALEN expression vector that specifically recognizes the pig miR-192 was constructed and then monoclonal epithelial cells defective in miR-192 were established. We found that miR-192 knockout led to enhance the adhesion ability of the E. coli strains F18ab, F18ac and K88ac, meanwhile increase the expression of target genes ( DLG5 and ALCAM ) by qPCR and western blotting analysis. The results suggested that miR-192 and its key target genes ( DLG5 and ALCAM ) could have a key role in E. coli infection. Based on our findings, we propose that further investigation of miR-192 function is likely to lead to insights into the molecular mechanisms of E. coli infection.