Surgical resection currently remains the mainstay of treatment for patients with gliomas of any grade. The maximum extent of surgical resection is associated with a long-term disease control; however, maximal resection of the brain tumor possibly results in additional neurological deficits. Therefore, improving the precision in brain tumor surgery by visual identification and screening of tumor cells can help to tackle this devastating disease. In the present study, BV2 microglial cells were engineered by iron oxide-nanoparticle stimulation as intraoperative optical imaging agent vehicles and loaded with near-infrared fluorescent dye DiD (DiDBV2-Fe) potentially for fluorescence-guided brain tumor surgery. Activation of BV2 microglial cells by citrate-stabilized iron oxide nanoparticles at a concentration of 62.5 μg mL-1 significantly inhibited M2 markers (arginase-1 and CD206), which is able to minimize risks of the immunosuppressive effects caused by the M2-like phenotype of microglial cells. Meanwhile, activated BV2 microglial cells showed up-regulation of arylsulfatase A, apolipoprotein E, transferrin, and ferritin heavy chain-1 gene expression that tends to promote microglia transport across the blood-brain barrier (BBB). Compared to DiDBV2 without iron oxide activation, DiDBV2-Fe indicated strong tumor tropism in response to monocyte chemoattractant protein-1 (CCL2) secreted by U87MG tumor cells. In vivo experiments proved that DiDBV2-Fe efficiently crossed the BBB and more than 90% fluorescence intensity generated by activated microglial cells was detected in the brain when administered through the carotid artery in an orthotopic glioblastoma mouse model. Notably, DiDBV2-Fe produced clear tumor border demarcation on near-infrared imaging and exhibited a superior tumor-to-brain fluorescence ratio to commercial 5-aminolevulinic acid. Accumulated DiDBV2-Fe induced a strong fluorescence signal in brain tumor tissue for a prolonged period (4-24 h), which is beneficial to perform complex and time-consuming brain operations. Overall, our study suggests that this newly engineered microglial cell has promise for enabling more accurate brain tumor imaging for fluorescence-guided resections.