This paper describes ambient vibration based seismic evaluation procedure of an isolated highway bridge. The procedure includes finite element modeling, ambient vibration testing, finite element model updating and time history analysis. Gulburnu Highway Bridge located on the Giresun-Espiye state highway is selected as a case study. Three dimensional finite element model of the bridge is created by SAP2000 software to determine the dynamic characteristics analytically. Since input force is not measured, Operational Modal Analysis is applied to identify dynamic characteristics. Enhanced Frequency Domain Decomposition and Stochastic Subspace Identification methods are used to obtain experimental dynamic characteristics. Analytical and experimental dynamic characteristic are compared with each other and finite element model of the bridge is updated by changing of material properties to reduce the differences between the results. Analytical model of the bridge after model updating is analyzed using 1992 Erzincan earthquake record to determine the seismic behavior. EW, NS and UP components of the ground motion are applied to the bridge at the longitudinal, transverse and vertical directions, respectively. It is demonstrated that the ambient vibration measurements are enough to identify the most significant modes of highway bridges. Maximum differences between the natural frequencies are reduced averagely from 9% to 2% by model updating. It is seen from the earthquake analyses that friction pendulum isolators are very effective in reducing the displacements and internal forces. (C) 2011 Elsevier Ltd. All rights reserved.