Metabolomic analyses in epidemiological studies have demonstrated a strong sexual dimorphism for most metabolites. Cross-sex hormone treatment (CSH) in transgender individuals enables the study of metabolites in a cross-gender setting. Targeted metabolomic profiling of serum of fasting transmen and transwomen at baseline and following 12 months of CSH (N = 20/group) was performed. Changes in 186 serum metabolites and metabolite ratios were determined by targeted metabolomics analysis based on ESI-LC-MS/MS. RandomForest (RF) analysis was applied to detect metabolites of highest interest for grouping of transwomen and transmen before and after initiation of CSH. Principal component analysis (PCA) was performed to check whether group differentiation was achievable according to these variables and to see if changes in metabolite levels could be explained by a priori gender differences. PCA predicted grouping of individuals-determined by the citrulline/arginine-ratio and the amino acids lysine, alanine and asymmetric dimethylarginine - in addition to the expected grouping due to changes in sex steroids and body composition. The fact that most of the investigated metabolites did, however, not change, indicates that the majority of sex dependent differences in metabolites reported in the literature before may primarily not be attributable to sex hormones but to other gender-differences.