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Background Magnetic targeting therapy may be a new method for the treatment of malignent tumors. The purpose of this study was to investigate the localization and distribution of ferrofluid microsphere of human serum albumin methotrexate (FM-HSA-MTX) carriers in the brain and to explore the magnetic targeting chemotherapy for malignant brain tumor. Methods Ninety SD rats were divided into three groups: targeting group, non-magnetic targeting group, and control group. Synthesized FM-HSA-MTX carriers (MTX 25 mg/kg) were injected into the systemic circulation via the caudal vein (magnetic targeting group, n=30). A 0.6 T magnetic field was placed around the right hemisphere. The non-magnetic targeting group (n=30) was administered with FM-HSA-MTX without external magnetic field, meanwhile the control group (n=30) was treated with MTX and a magnetic field. Random serial sacrifices (n=10) were conducted at 15, 30 and 45 minutes after drug administration. Bilateral hemispheres were collected respectively, and analyzed for total MTX content. Results MTX content in the right hemisphere of the magnetic targeting group was significantly higher than that in the other two groups at 15, 30 and 45 minutes after drug administration (P＜0.05) No difference was seen between the non-targeting group and control group. In the magnetic targeting group, MTX returned to the peak level [(0.564±0.018) mg/g, q15-45=32.252, P＜0.05] 45 minutes after the injection but it deceased in the other two groups [non-magnetic targeting group: (0.060±0.015) mg/g, q15-45=9.245, P＜0.05, control group: (0.074±0.045) mg/g, q15-45=6.299, P＜0.05]. In the magnetic targeting group, the concentration of MTX in the right hemisphere was significantly higher than that in the left hemisphere (t45min=21.135, P=0.000) but no difference was observed between bilateral hemispheres in the other two groups (non-magnetic targeting group: t45min=0.434, P=0.670; control group: t45min=0.533, P=0.600). Conclusion In the presence of the external magnetic field, FM-HSA-MTX can distribute successfully in the targeting areas of the brain.