The aberrant activation of osteoblasts in the early stage is one of the critical steps during the pathogenesis of skeletal fluorosis. The endoplasmic reticulum (ER) stresses and unfolded protein response (UPR) are initiated to alleviate the accumulation of unfolded proteins against cell injury. The previous researches had demonstrated that fluoride induced ER stress in other cells or tissues. In this study, we determined the ER stress and UPR to investigate their roles in aberrant activation of fluoride-treated osteoblasts. The gene expression of bone markers and UPR factors in MC3T3-E1 cells treated with varying doses of fluoride administration was analyzed. Meantime, levels of glutathione and glutathione disulfide were tested by the ultraperformance liquid chromatography-tandem mass spectrometry applications. Our results indicated that a certain dose and period of fluoride administration induced cell proliferation and differentiation, and Runx2 was involved in the regulation of osteoblastic differentiation of MC3T3-E1 cells. Increase trend of Runx2 expression was consistent with change of marker of ER stress. Fluoride caused ER stress and stimulated UPR during the process of osteoblast maturation, while oxidative stress was also active in the occurrence of ER stress. These data indicated that ER stress and UPR were possibly involved in the action of fluoride on osteoblasts.