Display omitted •Metabolomics and molecular pharmacology were combined performed for analysis.•Apoptosis-related metabolic pathways were found through metabolomics.•Gei Herba can mediate mitochondrial, PI3K/Akt and JAK/STAT pathways in mice. Gei Herba, Chinese named Lanbuzheng (LBZ), is a traditional Chinese medicine promotes hematopoiesis, yet the underlying mechanism for this effect remains largely unknown. In the present study, a novel approach combining LC–MS metabolomics and molecular pharmacology was developed to investigate the hematopoietic effect and mechanism of LBZ on hematopoietic dysfunction (HD) caused by cyclophosphamide (CTX) in treated mice. The results show that LBZ can reduce damage in the spleen, a result consistent with the peripheral hemogram. Fourteen potential biomarkers were identified in the spleen by metabolic profiles analysis, including 5-hydroxymethyluracil, ascorbalamic acid, adenosine 5′-monophosphate, menadiol disulfate, l-homocysteine sulfonic acid and l-carnitine. Change in biomarker levels suggest that LBZ mainly affects β-oxidation of very-long-chain fatty acids, oxidation of branched chain fatty acids and carnitine synthesis, and those metabolites produced along with related metabolic pathways are closely associated with anti-apoptosis. A molecular pharmacology approach was simultaneously developed to examine accompanying cellular signaling mechanisms. LBZ activates PI3K/Akt signaling pathways and granulocyte-colony-stimulating-factor (G-CSF)-mediated Janus kinase 2 (JAK2)/transcription 3 (STAT3), resulting in inhibiting the release of cytochrome c. Further, LBZ inhibits caspase-mediated mitochondrial-dependent apoptosis mediated by caspase-9 and caspase-3. LBZ can thus reduce CTX-induced HD via G-CSF-mediated JAK2/STAT3 signaling and PI3K/Akt mitochondrial-dependent apoptotic pathways. The present study combines metabolomic and molecular pharmacological methods to elucidate mechanisms for the protective effect of LBZ on mouse HD following CTX-induced damage. This approach may be useful for exploring mechanisms of action of other drugs.