Docetaxel (DTX) is a highly effective anti-tumor drug frequently used in clinical practice. Previous reports indicated that complications after DTX therapy could be related to retinal pigment epithelial (RPE) cell dysfunction, although no direct reports of this relationship have been published. In this study, human embryonic stem cell–derived RPE (hESC-RPE) cells were used to explore the effects of DTX on their morphology, viability, apoptosis, proliferation, and cell cycle. We also searched for DTX residue in these cells. DTX had a time- and concentration-dependent inhibitory effect on hESC-RPE cell viability, and the cells only survived after 24 h of stimulation with 0.1 mg/mL of DTX. Following drug withdrawal, the cell morphology continued to change, and hESC-RPE cell damage was observed. High-performance liquid chromatography/mass spectrometry showed that some unmetabolized DTX remained in hESC-RPE cells after the 48 and 120 h DTX treatments. Flow cytometry and immunofluorescence revealed that DTX significantly enhanced apoptosis, and the Cell Counting Kit-8 assay and flow cytometry indicated that DTX inhibited cell proliferation and blocked the cell cycle. These results suggest that DTX has a direct cytotoxic effect on hESC-RPE cells. Thus, RPE cell damage after DTX treatment may present an important safety problem that could potentially limit the application of this drug in clinical practice. The findings of this study suggest that clinicians should weigh the benefits of DTX versus the risks of ocular adverse reactions rationally. Timely diagnostic evaluation and drug withdrawal will be conducive to the recovery of patients' visual acuity.