The goal of this study was to screen bioactive peptides to identify an efficient antagonist of multiple pro-inflammatory chemokines that inhibits the pathological process of inflammatory diseases. A phage display library was sequentially screened by binding phages. The binding properties of individual phage clones to LPS-activated PBMCs were determined using cell-based ELISAs. The positive clones were selected and determined by chemotaxis assays. A high-activity peptide was determined to inhibit carrageenan-induced paw oedema and formaldehyde-induced arthritis in Wistar rats in vivo. A possible mechanism of inflammation inhibition involving chemokine mRNA by the peptide was determined by analyzing mRNA expression levels of chemokines and tristetraprolin (TTP) by SqRT-PCR.Nineteen phage clones were selected after four rounds of biopanning with a cut-off of 3-fold higher binding to LPS-activated PBMCs than to normal PBMCs. Nine of the phage clones inhibited IL-8, MCP-1, and MIP-1β production in vitro. Five clones displayed the same peptide(CI-S5)most robustly inhibited the chemotactic activity in vitro and reduced paw oedema and arthritis in Wistar rats in vivo. SqRT-PCR results indicated that mRNA expression of IL-8, MCP-1, and MIP-1β were reduced and TTP mRNA expression was increased in the CI-S5 treatment group. Our data demonstrate that CI-S5 is a broad-spectrum antagonist of pro-inflammatory chemokines as it enhances the expression of TTP to reduce chemokine mRNA expression. This study provides a basis for the development of new peptide-based therapies for the treatment of inflammatory diseases.