Organic compounds have been linked to adverse pregnancy complications. Perfluorooctanesulfonic acid (PFOS), a man-made fluorosurfactant and global pollutant, has been shown to induce oxidative stress in various cell types. Oxidative stress plays a key role in leading several placental diseases including preeclampsia (PE), gestational diabetes, spontaneous abortion, preterm labor, and intrauterine growth restriction. Recently, epigenetic regulation such as histone modifications, DNA methylation, and microRNAs (miRNAs), are shown to be associated with oxidative stress as well as pregnancy complications such as PE. However, whether PFOS exerts its detrimental effects in the placenta through epigenetics remains to be unveiled. Therefore, we aimed to investigate the effect of PFOS-induced reactive oxygen species (ROS) generation in first trimester human trophoblast cell line (HTR-8/SVneo) and whether epigenetic regulation is involved in this process. When treated with a range of PFOS doses at 24 and 48 h, even at 10 μM, it significantly increased the ROS production and decreased gene and protein expression, respectively, of the DNA methyltransferases DNMT1 (p < 0.001; p < 0.05), DNMT3A (p < 0.001; p < 0.05), and DNMT3B (p < 0.01; p < 0.01) and the sirtuins, for example, SIRT1 (p < 0.001; p < 0.001) and SIRT3 (p < 0.001; p < 0.05), while reducing global DNA methylation (p < 0.01) and increasing protein lysine acetylation (p < 0.001) as compared to vehicle controls. Interestingly, PFOS (10 μM) significantly increased miR29-b (p < 0.01), which has been previously reported to be associated with PE. The observed epigenetic effects were shown to be dependent on the expression of miR-29b, as knockdown of miR-29b significantly alters the gene and protein expression of DNMT1, DNMT3A, DNMT3B, SIRT1, and SIRT3 and ROS production as well as global DNA methylation and protein acetylation. This study provides for the first time a novel insight into PFOS-induced ROS generation via regulation of sets of the interactive epigenetic circuit in the placenta, which may lead to pregnancy complications.