Anthracnose caused by Colletotrichum lupini is an essential problem limiting lupine cultivation. In this study, we analyzed metabolic profiles of two white lupin cultivars with different susceptibility to primary infection caused by two C. lupini strains isolated from a single Russian field to reveal key metabolites and metabolic pathways involved in the defense response of seedlings to the pathogen. Multivariate and hierarchical clustering analyses showed that anthracnose-resistant cultivar SN 5–19 had a more pronounced defense response against the infection than the sensitive Michurinskij cultivar. Volcano plot analysis indicated fumaric acid and GABA as potential markers of C. lupini infection. The pathway analyses revealed that the metabolic alterations are conserved across susceptible and tolerant cultivars, indicating the importance of nitrogen mobilization in response to C. lupini infections.