The effect of sex as a modifier of cellular function is under-investigated. Whether gender influences estrogen (E2)-mediated mitochondrial cytoprotection was determined using cell cultures of normal human lens epithelia (nHLE) from post-mortem male and female donors. Experimental indicators assessed included differences in estrogen receptor- (ER-) isoform expression, receptor localization in mitochondria and estrogen-mediated prevention of loss of mitochondrial membrane potential using the potentiometric fluorescent compound, JC-1, after nHLE were exposed to peroxide. The influence of wild-type estrogen receptor- (wtER-1) was also assessed using wtER-1-specific siRNA to suppress expression. A triple primer PCR assay was employed to determine the proportional distribution of the receptor isoforms (wtER-1, 2 and 5) from the total ER- message pool in male and female cell cultures. Irrespective of gender, nHLE express wtER-1 and the ER-2 and ER-5 splice variants in similar ratios. Confocal microscopy and immunofluorescence revealed localization of the wild-type receptor in peripheral mitochondrial arrays and perinuclear mitochondria, as well as nuclear staining in both cell populations. The ER-2 and ER-5 isoforms were distributed primarily in the nucleus and cytosol, respectively; no association with the mitochondria was detected. Both male and female nHLE treated with E2 (1 µM) displayed similar levels of protection against peroxide-induced oxidative stress. In conjunction with acute oxidative insult, RNA suppression of wtER-1 elicited the collapse of mitochondrial membrane potential and markedly diminished the otherwise protective effects of E2. Thus, while the estrogen-mediated prevention of mitochondrial membrane permeability transition is gender-independent, the mechanism of estrogen-induced mitochondrial cytoprotection is wtER-1-dependent.