Abstract Mesenchymal stem cells (MSCs) and macrophages collaboratively contribute to bone regeneration after injury. However, the detailed mechanisms underlying the interaction between MSCs and inflammatory macrophages (M1) remain unclear. A macrophage-depleted tooth extraction model was generated in 5-weeks-old, female C57BL/6 J mice using clodronate liposome (12.5 mg/kg/mouse, intraperitoneally) or saline injection (control) prior to maxillary first molar extraction. The mice were sacrificed on days 1, 3, 5, 7, and 10 after tooth extraction (n = 4). Regenerated bone volume evaluation of tooth extraction socket (TES) and histochemical analysis of CD80+M1, CD206+M2 (anti-inflammatory macrophages), PDGFRα+MSC, and TNF-α+ cells were performed. In vitro, isolated MSCs with or without TNF-α-stimulation (10 ng/ml, 24 hour, n = 3) were bulk RNA-sequenced (RNA-Seq) to identify TNF-α stimulation-specific MSC transcriptomes. Day7 micro-CT and HE staining revealed significantly lower mean bone volume (clodronate vs control:0.01 mm3 vs. 0.02 mm3, p < 0.0001) and mean percentage of regenerated bone area per total TES in clodronate group (41.97% vs. 54.03%, p < 0.0001). Chlodronate group showed significant reduction in mean number of CD80+, TNF-α+, PDGFRα+ and CD80+TNF-α+ cells on day5 (306.5 vs. 558.8, p < 0.0001; 280.5 vs. 543.8, p < 0.0001; 365.0 vs. 633.0, p < 0.0001, 29.0 vs. 42.5, p < 0.0001), while these cells recovered significantly on day7 (493.3 vs. 396.0, p = 0.0004; 479.3 vs. 384.5, p = 0.0008; 593.0 vs. 473.0, p = 0.0010, 41.0 vs. 32.5, p = 0.0003). RNA-Seq analysis showed that 15 genes (|log2FC| > 5.0, log2TPM > 5) after TNF-α-stimulation were candidates for regulating MSC’s immunomodulatory capacity. In vivo, Clec4e and Gbp6 are involved in inflammation and bone formation. Clec4e, Gbp6, and Cxcl10 knockdown increased osteogenic differentiation of MSCs in vitro. Temporal reduction followed by apparent recovery of TNF-α-producing M1 macrophages and MSCs after temporal macrophage depletion suggests that TNF-α activated MSCs during TES healing. In vitro mimicking of the effect of TNF-α on MSCs indicated that there are 15 candidate MSC genes for regulation of immunomodulatory capacity.