The p38 mitogen-activated protein kinase (MAPK) signaling pathway is aberrantly expressed and maintains transformed cell growth in malignant human cholangiocytes. Because cell growth requires and is intimately related to protein synthesis, our aims were to assess the effect of p38 MAPK signaling on protein synthesis during growth of malignant human cholangiocytes. Inhibition of p38 MAPK activity during mitogenic stimulation decreased protein synthesis rates and tumor cell xenograft growth in nude mice. Altered protein synthesis resulted from decreased translational efficiency with impaired initiation of translation. Mitogenic stimulation resulted in phosphorylation of the eukaryotic initiation factor (eIF)-4E. Inhibition of p38 MAPK signaling or functional dysregulation of translation by small interfering double-stranded RNA (siRNA) to eIF-4E decreased anchorage-independent growth of malignant cholangiocytes. In conclusion, these studies identify a relationship between p38 MAPK activity and the regulation of protein synthesis during human cholangiocarcinoma growth. As protein synthesis is intimately linked to cell growth, dysregulation of translation initiation is a mechanism by which cellular p38 MAPK signaling participates in growth regulation of malignant cholangiocytes.