Plant small-RNAs regulate various biological processes by manipulating the expression of target genes at the transcriptional and post-transcriptional levels. However, little is known about the response and the functional roles of sRNAs, particularly small-interfering RNAs (siRNAs), in the soybean-soybean cyst nematode interaction. In this study, siRNA data from 24 sRNA libraries constructed from SCN-infected and non-SCN-infected resistant and susceptible soybean roots were analysed in silico. A total of 26 novel siRNAs including 17 phasiRNAs and 9 nat-siRNAs, as well as two phasiRNAs that were differentially expressed (DE)in three comparisons, were identified. Then, using qRT-PCR, the expression of majority of siRNAs was found to be downregulated after SCN infection, and the expression patterns of DE siRNAs were confirmed. Further functional annotation analyses revealed that the target genes of these siRNA were highly related to disease resistance, which included the genes coding for the NB-ARC domain, leucine-rich repeats, and Hs1pro-1 homologous proteins. Overall, the present research identified novel siRNAs and annotated their target genes, thereby laying the foundation for deciphering the roles of siRNAs in the soybean-SCN interaction.