Flower longevity is one of the most important traits in ornamental plants. In Japanese morning glory (Ipomoea nil), EPHEMERAL1 (EPH1), a NAC transcription factor, is reportedly a key regulator of petal senescence. CRISPR/Cas9-mediated targeted mutagenesis is a powerful tool for crop breeding as well as for biological research. Here we report the application of CRISPR/Cas9 technology to targeted mutagenesis of the EPH1 gene in I. nil. Three regions within the EPH1 gene were simultaneously targeted by a single binary vector containing three single-guide RNA cassettes. We selected eight T transgenic plants containing the transferred DNA (T-DNA). Cleaved amplified polymorphic sequence (CAPS) analysis revealed that mutations occurred at single or multiple target sites in all eight plants. These plants harbored various mutations consisting of single base insertions and/or deletions of a single or more than two bases at the target sites. Several mutations generated at target sites were inherited in the T progeny with or without T-DNA insertions. Mutant plants in the T generations exhibited a clear delay in petal senescence. These results confirm that CRISPR/Cas9 technology can efficiently induce mutations in a target I. nil gene and that EPH1 plays a crucial role in the regulation of petal senescence. The eph1 mutants obtained in this study will be a useful tool for the elucidation of regulatory mechanisms in petal senescence.