Formaldehyde that leaches out of formaldehyde-releasing root canal sealers, specifically from setting material extruded into the periapical region may participate in the development of periapical inflammation or the continuation of a pre-existing periapical lesion. However, the effects of formaldehyde on human osteoblasts have not been investigated. The aim of this study was to evaluate the mechanisms of cytotoxicity of formaldehyde on human osteoblastic cell line U2OS in vitro. Cytotoxicity and cell proliferation assays were performed to elucidate the adverse effects of formaldehyde on U2OS cells. Formaldehyde demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (p<0.05). The 50% inhibition concentration of formaldehyde was about 3 mM. Formaldehyde also inhibited cell proliferation during a 3-day culture period (p<0.05). To determine whether glutathione (GSH) levels were important in the cytotoxicity of formaldehyde, we pretreated cells with the GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. The addition of OTZ acted as a protective effect on the formaldehyde-induced cytotoxicity (p<0.05). In contrast, the addition of BSO enhanced the formaldehyde-induced cytotoxicity (p<0.05). Taken together, the levels of formaldehyde tested inhibited cell growth and proliferation on U2OS cells. Formaldehyde has significant potential for periapical toxicity. These inhibitory effects were associated with intracellular GSH levels.