Background: Soybean cyst nematode Heterodera glycines is one of the most devastating pathogens on soybean and causes severe annual yield loss worldwide. Different soybean varieties exhibit different responses to soybean cyst nematode infection at various levels, such as genomic, transcriptional, proteomic, and metabolomic levels. However, there were not yet any reports on the differential responses of resistant and susceptible soybeans infected with soybean cyst nematode by combining the metabolomic analyses and transcriptomics.Results: In this study, a highly-resistant variety PI 437654 and three susceptible varieties Williams 82, Zhonghuang 13 and Hefeng 47 were used as the test materials to clarify the differences in metabolites and transcriptomics between resistant and susceptible soybeans before and after SCN infection. A local metabolite-calibrated database was used to identify potential differential metabolites, and the differences of metabolites and metabolic pathways were compared between the resistant and susceptible soybean varieties after inoculation with SCN. Totally, 37 differential metabolites and 20 KEGG metabolic pathways were identified, which were divided into three categories including the metabolites/pathways overlapped among resistant and susceptible soybeans, specific in susceptible or resistant soybeans, respectively. Twelve differential metabolites were found to be involved in predicted KEGG metabolite pathways. Moreover, 14 specifically differential metabolites such as significantly up-regulated nicotine and down-regulated D-aspartic acid, and their involved KEGG pathways such as tropane, piperidine and pyridine alkaloid biosynthesis, alanine, aspartate and glutamate metabolisms, sphingolipid metabolism and arginine biosynthesis were significantly changed and abundantly enriched in the resistant soybean, and likely played pivotal roles in defensing against SCN infection. Three key metabolites including N-acetyltranexamic acid, nicotine, and D, L-typotophan, which were found to be significantly up-regulated in the resistant soybean PI437654 infected by SCN, classified into two types and used for combination analyses with the transcriptomic expression profiling. Associated genes were predicted, and suggested their likely biological processes, cellular component, molecular function and involved pathways. Conclusions: Our results not only found out the potential novel metabolites and associated genes in the resistant response of soybean to soybean cyst nematode, but also provided new insights into the interactions between soybean and soybean cyst nematode.