Hepcidin is a liver-expressed iron-regulating hormone that also is an antimicrobial peptide. Though the iron regulatory function of porcine hepcidin (pHepc) has been extensively investigated, the studies on the relationship between pHepc and bacteria are limited. The aim of current study was to evaluate its antibacterial activity against Escherichia coli K88 (E. coli K88) and investigate the effect of pHepc on bacterial infection in vitro. The antibacterial activity against pathogen bacteria was evaluated via radial diffusion, colony forming count, transmission electron microscopy (TEM) and DNA binding assays. Invasion assay and immunofluorescence microscopy were employed to determine its effect on bacterial infection. The results showed that pHepc exerted an iron-independent bacteriostatic activity against E. coli K88 in a dose-dependent manner. pHepc-treated E. coli K88 exhibited longer cells and cytoplasm unevenly distribution. To determine whether pHepc inhibited bacterial infection, piglet intestinal epithelial cells were challenged with pHepc-treated or untreated E. coli K88. The ability of pHepc-treated bacteria to invade IPEC-1 epithelial cells was impaired. pHepc significantly reduced 33.5% amount of E. coli K88 which adhered or invaded to cells (P < 0.05). Another human epithelial colorectal adenocarcinoma cell Caco-2 exhibited the same inhibition pattern by pHepc treatment (P < 0.05). By scanning electron microscopy (SEM), pHepc-treated E. coli K88 was aggregated, and higher magnification revealed a net-like meshwork of fibrils emanating from the bacterial surface that entangled the bacteria. It suggested that pHepc could afford protection against infection of E. coli K88 by a novel aggregation strategy, which will contribute broadly to piglet innate immunity.