Gemcitabine, 2'-deoxy-2', 2'-difluorocytidine (dFdC), is a drug approved for use against various solid tumors. Clinically, this moderately toxic nucleoside analog causes peripheral neuropathy, hematological dysfunction, and pulmonary toxicity in cancer patients. Although these side effects closely mimic symptoms of mitochondrial dysfunction, there is no direct evidence to show gemcitabine interferes with mitochondrial DNA replication catalyzed by human DNA polymerase gamma. Here we employed presteady state kinetic methods to directly investigate the incorporation of the 5'-triphosphorylated form of gemcitabine (dFdCTP), the excision of the incorporated monophosphorylated form (dFdCMP), and the bypass of template base dFdC catalyzed by human DNA polymerase gamma. Opposite template base dG, dFdCTP was incorporated with a 432-fold lower efficiency than dCTP. Although dFdC is not a chain terminator, the incorporated dFdCMP decreased the incorporation efficiency of the next 2 correct nucleotides by 214- and 7-fold, respectively. Moreover, the primer 3'-dFdCMP was excised with a 50-fold slower rate than the matched 3'-dCMP. When dFdC was encountered as a template base, DNA polymerase gamma paused at the lesion and one downstream position but eventually elongated the primer to full-length product. These pauses were because of a 1,000-fold decrease in nucleotide incorporation efficiency. Interestingly, the polymerase fidelity at these pause sites decreased by 2 orders of magnitude. Thus, our pre-steady state kinetic studies provide direct evidence demonstrating the inhibitory effect of gemcitabine on the activity of human mitochondrial DNA polymerase.