Benzene is identified as a carcinogen. Continued exposure of benzene may eventually lead to damage to the bone marrow, accompanied by pancytopenia, aplastic anemia or leukemia. This paper explores the variations of endogenous metabolites to provide possible clues for the molecular mechanism of benzene-induced hematotoxicity. Liquid chromatography coupled with time of flight-mass spectrometry (LC-TOF-MS) and principal component analysis (PCA) was applied to investigate the variation of endogenous metabolites in bone marrow cells and plasma of male C3H/He mice. The mice were injected subcutaneously with benzene (0, 300, 600 mg/day) once daily for seven days. The body weights, relative organ weights, blood parameters and bone marrow smears were also analyzed. The results indicated that benzene caused disturbances in the metabolism of oxidation of fatty acids and essential amino acids (lysine, phenylalanine and tyrosine) in bone marrow cells. Moreover, fatty acid oxidation was also disturbed in plasma and thus might be a common disturbed metabolic pathway induced by benzene in multiple organs. This study aims to investigate the underlying molecular mechanisms involved in benzene hematotoxicity, especially in bone marrow cells.