Soybean is an important crop worldwide, its production is limited by soybean stem canker disease caused by Diaporthe caulivora. In this study, two contrasting soybean cultivars, Williams (susceptible) and Génesis 5601 (resistant) were compared in response to infection with D.caulivora. Génesis 5601 was more resistant to fungal infection than Williams, evidenced by reduced length lesions, disease severity index and pathogen biomass. Transcriptional profiling was performed in D.caulivora-inoculated and control tissues. In total, 2322 and 1855 genes were differentially expressed in Génesis 5601 and Williams, respectively. Under basal conditions Génesis 5601 presents higher expression of genes related to plant defense. At 8 hours post inoculation an earlier defense response was activated in Génesis 5601, demonstrated by upregulation of 1028 compared to 434 genes in Williams. Resistance to D.caulivora was associated with perception of the pathogen by surface and intracellular receptors, and defense activation through transcription factors, biosynthesis of phenylpropanoids, hormones, small heat shock proteins and genes with different roles in defense. Moreover, cell wall modifications, accumulation of phenolic compounds and reactive oxygen species were observed in stems of inoculated plants compared to the control. These findings provide novel insights into soybean molecular defense developed to control this pathogen, and a foundation for improving resistance in breeding programs.