Fatty acid-binding protein (FABP) regulates polyunsaturated fatty acids intracellular trafficking and functions as a signal transduction via modulation of gene expression. We have demonstrated that FABP3 protein was observed in microglia of the median eminence (ME) of hypothalamus and this protein was increased in the ME of pain model mice. These changes were correlated with the increment of hypothalamic docosahexaenoic acid (DHA) levels. Here, we assessed the effect of DHA on FABP3 expression using MG6 cell, a microglia cell line. Also, we tested the effect of FABP inhibitor on the mechanical allodynia in postoperative pain model mice. MG6 cells were cultured in Dulbecco's modified eagle medium with or without 10% fetal bovine serum (FBS) as a cell stress. FABP3 was measured by qPCR. Mechanical allodynia was assessed by von Frey test. FABP3 mRNA was expressed on the MG6 cell. Under the condition of serum-free media, FABP3 mRNA was also significantly increased compared to the media with 10% FBS. This increment was suppressed by DHA (300 μM). Repeated intraventricular injection of FABP inhibitor was significantly suppressed mechanical allodynia in postoperative pain mice. These results indicated that DHA might be involved in the regulation of microglial FABP3, and brain FABP might work as a regulator of pain.