The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. To date, battling Phytophthora diseases is challenged by a lack of understanding on the pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat-inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in the miR393-knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from the canonical resistance genes encoding nucleotide binding-leucine rich repeat (NB-LRR) proteins and the genes encoding pentatricopeptide repeat (PPR)-containing proteins. This work highlights specific miRNAs and phasiRNAs that regulate defense-associated genes in soybean during Phytophthora infection. This article is protected by copyright. All rights reserved.