Valproic acid (VPA) is commonly used to treat bipolar disorder (BD), but its therapeutic role has not been clearly elucidated. To gain insights into VPA's mechanism of action, proteomic analysis was used to identify differentially expressed proteins in the rat prefrontal cortex (PFC), a region particularly affected in BD, after 6 weeks of VPA treatment. Proteins from PFCs of control and VPA-treated rats were separated by 2D-DIGE and identified by mass spectrometry. Among the 2,826 protein spots resolved, the abundance of 19 proteins was found to be significantly altered in the VPA-treated group (with the levels of three proteins increasing and 16 decreasing). Seven proteins whose levels were significantly altered after chronic VPA exposure were quantified by Western blot analysis. The 19 identified proteins represent potential new targets for VPA action and should aid in our understanding of the role of VPA in BD. © 2014 Wiley Periodicals, Inc.