Treatment of MLO-Y4 with 0.3 mM H2O2 induced apoptosis that was significantly inhibited (p ≤ 0.002) when the cells were pre-treated for 1 h with either 17β-estradiol, Raloxifene or LY117018 (10 nM). The stereoisomer 17α-estradiol also prevented H2O2 induced apoptosis in MLO-Y4. Importantly, pre-treatment of ER-negative HEK293 cells with either 1 μM, 100 nM or 10 nM 17β-estradiol, Raloxifene or LY117018 significantly inhibited H2O2 induced apoptosis in these cells (p ≤ 4.2 × 10− 5) indicating an estrogen receptor-independent effect of these compounds. Comparisons of 17β-estradiol and similar molecules containing the putative free radical scavenger C3-OH moiety on the steroid A-ring (17α-estradiol, 17α-ethinylestradiol; 10 nM) with structurally related molecules lacking the C3-OH grouping (Mestranol and Quinestrol; 10 nM) demonstrated that only compounds containing the C3-OH moiety showed anti-apoptotic behavior in these studies (p ≤ 0.0033). Similarly the identification of the presence of reactive oxygen species (ROS) in cells as evidenced by the free radical indicator 2′7′-dichlorodihydrofluorescein diacetate demonstrated that 17β-estradiol, SERMs and related molecules with C3-OH moiety were capable of blocking ROS generated in cells by H2O2 (p ≤ 0.002) while Mestranol and Quinestrol showed no such blockade. It is possible that the loss of osteocytes during estrogen insufficiency may occur through a failure to suppress the activity of naturally occurring or disease associated oxidant molecules. These data suggest that the osteocyte protective effects of estrogen and SERMs may operate through a common receptor-independent mechanism which may be related to the antioxidant activity of these molecules.