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Vine tea (Ampelopsis grossedentata) is a traditional herb widely consumed in southwestern China that possesses paramount potential for human health. In 2021, the outbreak of typical leaf blight disease was observed in almost all vine tea plantations in Zhangjiajie of Hunan province, resulting in significant economic losses of vine tea production. In this study, we identified Neopestalotiopsis clavispora as the causal agent of vine tea leaf blight via its morphological characteristics and molecular identification. The sensitivity distribution of N. clavispora isolates to picoxystrobin were determined based on mycelial growth and spore germination inhibition assays. The EC50 values for mycelial growth ranged from 0.0062 to 0.0658 µg/mL, with a mean of 0.0282 ± 0.0148 µg/mL. The EC50 values for spore germination ranged from 0.0014 to 0.0099 µg/mL, and the mean value was 0.0048 ± 0.0022 µg/mL. Picoxystrobin increased fungal cell membrane permeability, but inhibited fungal ATP biosynthesis. Moreover, picoxystrobin exhibited good in planta control efficacy on vine tea leaves. Three picoxystrobin-resistant mutants were obtained in the current study, but no mutations were detected in the N. clavispora Cytb gene. Competitive ability assays showed that the conidium production and pathogenicity of all picoxystrobin-resistant mutants decreased as compared to their progenitors, indicating that picoxystrobin-resistant mutants suffer fitness penalty. These findings provide important evidence for picoxystrobin in vine tea leaf blight management and increase understanding of the resistance mechanism of picoxystrobin against N. clavispora. [ABSTRACT FROM AUTHOR]