Objectives: pH-responsive zeolite imidazolate framework-8 (ZIF-8) enables selective release of 5-fluorouracil (5-FU) within the acidic tumor microenvironment. However, the direct effects of ZIF-8 itself on cancer cells or surrounding tissues remain unclear. Since oral cancer involves interactions between epithelial tumor cells and stromal cells, comparing the effects of ZIF-8 on epithelial cancer cells and orofacial mesenchymal stem/stromal cells (OMSCs) is critical to understanding its broader biological impact. Methods: The effects of ZIF-8 on SCC7 epithelial cancer cells and OMSCs, including periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs), were evaluated using RNA sequencing, nuclear staining, live/dead assays, and immunocytochemistry. Cells were treated with 0, 1, 10, or 100 μg/mL ZIF-8. Based on nuclear staining results, live/dead viability assays were conducted on SCC7 and DPSCs treated with 0 or 100 μg/mL ZIF-8. Apoptosis-related markers (Bax, caspase-3, caspase-6, and caspase-10) were assessed following exposure to 100 μg/mL ZIF-8. Results: Transcriptomic analysis revealed that ZIF-8 not only facilitates selective 5-FU release but also directly induces apoptosis in SCC7 cells compared with 5-FU alone. At 100 μg/mL ZIF-8, SCC7 viability was significantly reduced, whereas OMSC viability was preserved. Nonviable SCC7 cells increased markedly compared with controls, while DPSCs showed no significant change. Apoptosis-related signaling was also elevated in SCC7 cells compared with DPSCs. Conclusions: ZIF-8 at 100 μg/mL selectively inhibits SCC7 growth while sparing OMSC viability and apoptosis.