Ethidium bromide (EtBr) is an intercalating agent, which binds tightly to mitochondrial DNA (mtDNA) during replication, and so blocks the function of mitochondria. EtBr inserts itself between the stacked bases in double-stranded DNA and specifically inhibits mtDNA transcription and replication by deleting RNA primers required for initiating mtDNA replication. In this study, the authors wanted to examine whether blocking mtDNA replication with EtBr could change the expression of stenmness genes and the expression of the immuneregulator B7-H3 in prostate cancer cell lines in vitro. Both PC-3 and DU145 prostate cancer cell lines were treated with 50 and 500 ng/mL of EtBr for 2 weeks. There was no difference in growth between EtBr-treated and control cells after 1 week. A slightly slower growth was observed for both cell lines during the second week of culture with EtBr compared to controls. After 2 weeks of culture with EtBr both cell lines showed increased expression of the stemness-related genes ABCG2, Oct3/4, Nanog1/Nanogp8, and CD44. Concomitantly, a dose-dependent increase of B7-H3 protein expression in both cell lines was identified and verified by both flow cytometry and immunocytochemistry. In conclusion, blocking mtDNA replication by EtBr induces increased expression of stemness genes, such as Oct3/4, Nanog, CD44, and ABCG2, in addition to the immune regulator B7-H3 in PC-3 and DU145 prostate cancer cell lines. The findings indicate that mitochondrial function may be associated with stemness of cancer cells and/or maintenance of a cancer stem cell phenotype. The finding of increased B7-H3 expression may be associated with the immunosuppression of cancer cells.