Cytochrome b5 (CB5) proteins are small heme-binding proteins, that influence cytochrome P450 activity. While only one CB5 isoform is found in mammals, higher plants have several isoforms of these proteins. The roles of the many CB5 isoforms in plants remain unknown. We hypothesized that CB5 proteins support the cytochrome P450 enzymes of plant specialized metabolism and found CB5C from Arabidopsis thaliana to co-express with glucosinolate biosynthetic genes. We characterized the glucosinolate profiles of two T-DNA insertion mutants of CB5C, and found that long-chained aliphatic glucosinolates were reduced in one of the mutant lines - a phenotype that was exaggerated upon methyl-jasmonate treatment. These results support the hypothesis, that CB5C influences glucosinolate biosynthesis, however, the mode of action remains unknown. Furthermore, the mutants differed in their biomass response to methyl jasmonate treatment. Thereby, our results highlight the varying effects of T-DNA insertion sites, as the two analyzed alleles show different phenotypes.