Martynoside (MAR) is a bioactive glycoside of Rehmannia glutinosa, a traditional Chinese herb frequently prescribed for treating chemotherapy-induced pancytopenia. Despite its clinical usage in China for thousands of years, the mechanism of MAR’s hematopoietic activity and its impact on chemotherapy-induced antitumor activity are still unclear. Here, we showed that MAR protected ex vivo bone marrow cells from 5-fluorouracil (5-FU)-induced cell death and inflammation response by down-regulating the TNF signaling pathway, in which II1b was the most regulatory gene. Besides, using mouse models with melanoma and colon cancer, we further demonstrated that MAR had protective effects against 5-FU-induced myelosuppression in mice without compromising its antitumor activity. Our results showed that MAR increased the number of bone marrow nucleated cells (BMNCs) and the percentage of leukocyte and granulocytic populations in 5-FU-induced myelosuppressive mice, accompanied by an increase in numbers of circulating white blood cells and platelets. The transcriptome profile of BMNCs further showed that the mode of action of MAR might be associated with the increased survival of BMNCs and the improvement of the bone marrow microenvironment. In summary, we revealed the potential molecular mechanism of MAR to counteract 5-FU-induced bone marrow cytotoxicity both ex vivo and in vivo, and highlighted its potential clinical usage in cancer patients experiencing chemotherapy-induced multi-lineage myelosuppression.