Feto-placental Hofbauer cells (HBCs) are macrophages residing in placental stroma. They are generally described as anti-inflammatory M2 polarized cells, promoting tolerance and tissue remodeling. In certain pathologies, however, a possible phenotypical switch towards pro-inflammatory M1 macrophages has been proposed. The study aimed to determine if HBCs can acquire an M1 phenotype under pro-inflammatory conditions in vitro. HBCs were isolated from healthy human term placentas. Cells were cultivated upon addition of LPS and INF-γ or IL-4 and IL-13 to induce the M1 and M2 phenotype, respectively. Specific cell polarization markers and cytokines, associated with respective phenotypes, were investigated by flow cytometry and ELISA. THP-1 macrophages served as positive control. Pro-inflammatory stimuli reduced M2 markers CD163 and DC-SIGN, but did not induce M1 markers. TNF-α release was increased, but at the same time TGF-β and IL-10 release was upregulated, resembling in part the M2b sub-phenotype. Anti-inflammatory stimuli had no effect on HBC polarization. HBCs maintain their M2 phenotype in vitro despite inflammatory stimuli, which might represent a state of adaption and tolerance to avoid rejection of the semiallogeneic feto-placental unit.