Objective To observe the expression characteristics of green fluorescent protein (GFP)-positive myeloid cells (mainly monocytes, macrophages, and neutrophils) and their pattern of response to diphtheria toxin (DT) in Lyz2 internal ribosome entry site (IRES) DT receptor/enhanced GFP (DTREGFP) mice. Methods Transgenic Lyz2 IRES DTREGFP mice (6 ~ 8 weeks old) and C57BL/6J mice with the same genetic background were selected randomly and injected intraperitoneally with DT (25 ng/g) for 3 consecutive days. Bone marrow, peripheral blood, and spleen cells were separated and made into single-cell suspensions with GFP and CD11b as markers. Changes in the proportion of GFP⁺ cells to total myeloid cells (CD11b⁺) was detected in these tissues at different time points before and after drug administration, using flow cytometry. Results Peripheral blood CD11b⁺GFP⁺ cells accounted for (24.62 ± 5.655) % of CD11b⁺ cells before DT injection, and this proportion gradually decreased after injection and reached a minimum of (7.982 ± 2.729) % on the third day. The proportions in spleen and bone marrow were (13.73 ± 2.994) % and (65.23 ± 4.261) % before DT injection, respectively, decreased most significantly on the first day after injection to (3.468 ± 0.5862) % and (50.98 ± 7.957) %, respectively, and then gradually recovered. Conclusions DT effectively reduced the proportions of monocytes, macrophages, and neutrophils in Lyz2 IRES DTREGFP mice, but the pattern of change varied among tissues.