Biofilm formation is easily found in patients suffered from ventilator-associated pneumonia (VAP) in neonatal intensive care unit (NICU) and makes the VAP infections not only harder to be treated but easier to relapse. In order to find some novel ways to inhibit biofilm formation, this study describe a previously unrecognized role for the human umbilical cord mesenchymal stem cells (hUCMSCs). In addition to multiple differentiation, hUCMSCs have the ability to prevent the biofilms formation in vitro by secreting antibacterial peptides (LL-37 and hBD-2). This occurred whilePA27853 and hUCMSCs were cocultured, and the filtrated medium, which was the supernatant containing antibacterial peptides (5.9 ng/ml of LL-37, 1.77 ng/ml of hBD-2), and inhibited the growth of the bacterial biofilm on the surface of tracheal tube (2.5#, for preterm infant). Using microarrays, we were able to demonstrate that the antibacterial peptides from hUCMSC affected biofilm formation by downregulating the gene-encoded polysaccharide biosynthesis protein. In addition, in order to find out the most suitable concentration of hUCMSCs,was cocultured with eight-level concentrations of hUCMSCs, and we found that the concentration of LL-37 was positively correlated with the concentration of hUCMSCs. Meanwhile, the concentration of LL-37 became stable while the hUCMSC concentration reaches higher than 5 × 10 cells/ml. But the concentration of hBD-2 had no significant correlation with hUCMSCs. The collection of these stem cells is not only limited by ethics but also reduces host rejection. This makes it possible to use autologous hUCMSCs to treat neonatal VAP.