Calf Spleen Extractive Injection (CSEI), extracted from the spleen of healthy cows (within 24 hours of birth), is a small-peptide-enriched extraction and often used as an ancillary agent in cancer therapy. This study evaluated the hematopoietic function of CSEI and its underlying mechanisms, principally in CHRF, K562 cells, BMNCs and a mouse model of cyclophosphamide (CTX)-induced hematopoietic suppression. CSEI promoted the proliferation and differentiation of CHRF and K562 cells, activated hematopoietic- and proliferation-related factors RSK1p90, ELK1 and c-Myc, and facilitated the expression of differentiation- and maturation-related transcription factors GATA-1, GATA-2. In the mice with hematopoietic suppression, 3 weeks of CSEI administration enhanced the bodyweights and thymus indices, suppressed the spleen indices and strongly elevated the production of HSPCs, neutrophils and B cells in bone marrow, ameliorated bone marrow cellularity, and regulated the ratio of peripheral blood cells. Proteome profiling combined with ELISA revealed that CSEI regulated the levels of cytokines, especially G-CSF and its related factors, in the spleen and plasma. Additional data revealed that CSEI promoted phosphorylation of STAT3, which was stimulated by G-CSF in both mice spleen and cultured BMNCs. Taken together, CSEI has the potential to improve hematopoietic function via the G-CSF-mediated JAK2/STAT3 signaling pathway.