Basic experimental study.To clarify the role of macrophages (Mϕs) in the osteogenic differentiation of ligamentum flavum (LF) cells.Mϕs and secreted factors are involved in the regulation of cell osteogenic differentiation, and play an important role in the process of heterotopic ossification. Whether Mϕs are involved in the development of ossification of the ligamentum flavum (OLF) have not been reported.The expression of CD68+ Mϕs in ossified LF tissue was identified by immunohistochemical staining. THP-1 cells were polarized to M1 and M2, and identified by flow cytometry and immunofluorescence. The alkaline phosphatase activity and osteogenic differentiation-related gene expression in LF cells were evaluated following incubation with each Mϕs conditioned medium (CM). ELISA was used to detect the pro-inflammatory cytokines in the supernatants, and qPCR was used to detect the expression of the corresponding receptors in the LF cells after incubation with the CM. LF cells were induced with CM-M1 in the presence of neutralizing antibodies to further test whether cytokines secreted by M1 Mϕs impacted their osteogenic differentiation.CD68+ Mϕs were found on the OLF samples. THP-1 cells were polarized into M1 and M2, and both M1 and M2 Mϕs promoted the osteogenic differentiation of LF cells. The concentrations of TNF-α, IL-1β, and IL-6 in M1 Mϕ supernatants were greater than those in M2, and greater levels of these cytokine receptors were observed in LF cells induced with CM-M1 than those with CM-M2. Osteogenic differentiation of LF cells induced by CM-M1 decreased after IL-6 was neutralized; however, not after IL-1β and TNF-α were neutralized.M1 Mϕ-derived IL-6 promotes the osteogenic differentiation of LF cells, which may be a pathway in which Mϕs regulate the osteogenic differentiation of LF cells.Level of Evidence: N/A.Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.