This paper addresses the ambient vibration-based finite element model updating of long span reinforced concrete highway bridges. The procedure includes ambient vibration tests under operational conditions, finite element modeling using special software and finite element model updating using some uncertain parameters such as section properties, damage, boundary conditions and material properties. The structural carrier system of the bridge consists of two main parts: arch and beam compartments. In this paper, the beam compartment is investigated. Three-dimensional finite element model of the beam compartment of the bridge is constituted using SAP2000 to determine the dynamic characteristics. Operational modal analysis (OMA) is performed for experimental measurements. Enhanced frequency domain decomposition (EFDD) method is used to extract dynamic characteristics. Analytical and experimentally identified dynamic characteristics are compared with another and finite element model of the beam compartment of the bridge is updated by changing some uncertain parameters such as section properties, damage, boundary conditions and material properties to reduce the differences between the results. It is demonstrated that the ambient vibration measurements are enough to identify the most significant modes of long span highway bridges. Maximum differences between the natural frequencies reduce in average from 46.7 to 2.39 % by model updating. In addition, a good conformity is found between mode shapes after model updating.