Nucleoside analogue drugs are widely used in cancer therapy and antiviral therapy, while fast metabolism, drug resistance, and severe side effects significantly limit their clinical applications. To address these issues, a variety of ester- and amide-linked prodrugs and their nanoparticulate formulations have been devised. However, most of these prodrugs suffer from inefficient transformation to native drugs in tumor. Here, we report an approach to conjugate gemcitabine, a kind of anticancer nucleoside drug and widely used to treat cancers, to polyketal backbone via pH-sensitive ketal linkage, and prepared gemcitabine-containing polyketal prodrug nanoparticles with minimal drug release under physiological conditions and acid-triggerable release of native gemcitabine. Intracellular and intratumoral degradation of the pH-sensitive gemcitabine-containing polyketal prodrug and incorporation of gemcitabine into DNA were confirmed by confocal microscopy using EdU, an analogue of gemcitabine. One single intravenous injection of these gemcitabine-containing polyketal prodrug nanoparticles demonstrated notable anticancer efficacy in the A2780 ovarian xenograft tumor model with increased survival rate and good safety. Our approach can be adopted for other diol nucleoside analogues to synthesize pH-sensitive nucleoside-polyketal prodrugs for developing anticancer and antiviral formulations.