FeO nanoparticles are the most widely used magnetic nanoparticles in the biomedicine field. The biodistribution of most nanoparticles in vivo is determined by the capture of macrophages; however, the effects of nanoparticles on macrophages remain poorly understood. Here, we demonstrated that FeO nanoparticles could reduce macrophage viability after 48 h of treatment and induce a shift in macrophage polarization toward the M1 phenotype; RNA sequencing revealed the activation of the ferroptosis pathway and upregulation compared to the control group. The expression in p53, xCT, glutathione peroxidase 4 (GPX4), and transferrin receptor (TFR) in macrophages was similar to that in erastin-induced ferroptosis in macrophages, and the ultrastructural morphology of mitochondria was consistent with that of erastin-treated cells. We used DCFH-DA to estimate the intracellular reactive oxygen species content in FeO nanoparticles treated with Ana-1 and JC-1 fluorescent probes to detect the mitochondrial membrane potential change; both showed to be time-dependent. Fer-1 inhibited the reduction of the glutathione/oxidized glutathione (GSH/GSSG) ratio and inhibited intracellular oxidative stress states; therefore, FeO nanoparticles induced ferroptosis in macrophages. Finally, we used pifithrin-α hydrobromide (PFT) as a p53 inhibitor to verify whether the high expression of p53 is involved in mediating this process. After PFT treatment, the live/dead cell rate, TFR, p53 expression, and GPX4 consumption were inhibited and mitigated the GSH/GSSG ratio reduction as well. This indicates that p53 may contribute to FeO nanoparticle-induced ferroptosis of macrophages. We provide a theoretical basis for the molecular mechanisms of ferroptosis in macrophages and the biotoxicity in vivo induced by FeO nanoparticles.