Set SeparatorImageUrl
<%@ Page Language="C#" %> <!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> <html xmlns="http://www.w3.org/1999/xhtml" > <head runat="server"> <title>Test Menu</title> </head> <body> <form id="form1" runat="server"> <div id="container"> <asp:Menu id="menuMain" runat="server"> <Items> <asp:MenuItem Text="Home" NavigateUrl="home.aspx" ImageUrl="images/menuHome.gif" SeparatorImageUrl="images/separator.gif" ToolTip="Click to return to home page" /> <asp:MenuItem Text="Catalog" PopOutImageUrl="images/menuPopout.gif" ImageUrl="images/menuCatalog.gif" SeparatorImageUrl="images/separator.gif"> <asp:MenuItem Text="Categories" NavigateUrl="categories.aspx" SeparatorImageUrl="images/separator.gif" ToolTip="View our books by category" /> <asp:MenuItem Text="Series" NavigateUrl="series.aspx" ToolTip="View our books by series"/> </asp:MenuItem> <asp:MenuItem Text="About Us" NavigateUrl="about.aspx" ImageUrl="images/menuABout.gif" ToolTip="Our history and contact info" /> </Items> </asp:Menu> <asp:SiteMapDataSource ID="siteSource" runat="server" ShowStartingNode="false" /> </div> </form> </body> </html>