Seed treatment with fungicides has been regarded as a principal, effective, and economic technique for soybean [ Glycine max (L.) Merr.] against pathogenic microorganisms during seed germination and seedling growth. Investigation of the characteristics of seed-treatment reagents is an indispensable basis for their application. The aim of the present work is to evaluate the use of pyraclostrobin as an ingredient for soybean seed treatment by investigating its accumulation−dissipation kinetics in plants, plant-growth activation, and protection against Phytophthora sojae. The results showed that the pyraclostrobin stimulated the visible growth (root and shoot length) of soybean plants, increased the chlorophyll level and root activity, and lowered the malonaldehyde (MDA) level. The peak level and bioavailability of pyraclostrobin in soybean roots were 19.9-and 33.2-fold those in leaves, respectively, indicating that pyraclostrobin was mainly accumulated in roots. Pyraclostrobin had a continuous positive effect on the flavonoid levels and the phenylalanine ammonialyase (PAL) activity in roots and leaves, which could enhance the plant defense system. Pyraclostrobin showed in vitro toxicity to P. sojae with a half-inhibition concentration (EC 50 ) of 1.59 and 1.24 μg/mL for pyraclostrobin and pyraclostrobin plus salicylhydroxamic acid (SHAM, an inhibitor of the alternative pathway of respiration), respectively. Seed treatment with pyraclostrobin significantly reduced the severity of Phytophthora root rot, with a control efficacy of 60.7%. To the best of our knowledge, this is the first report on the characteristics of pyraclostrobin used in soybean seed treatment and its efficacy against Phytophthora root rot.