Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here， a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T-T generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1， PR12， and PAL， jasmonic acid-dependent gene PPO， and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover， the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL)， polyphenoloxidase (PPO)， peroxidase， and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways， thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.