Introduction: Sex steroid hormones exert a wide range of effects on metabolism. New techniques such as metabolomic profiling allow for a deeper insight into metabolic regulation. In epidemiological samples it has been demonstrated that most of these metabolites show sex-specific differences. However, if these differences are attributable to the effects of sex hormones or genetics is little understood so far. Methods: We performed targeted metabolomics measurements in serum of fasting transmen (F2 M) and transwomen (M2F) at baseline and following 12 months of cross-sex hormone treatment (N = 20/group). Subjects investigated in this study were part of the European Network for the Investigation of Gender Incongruence (ENIGI). The metabolites were quantified by ESI-LC-MS/MS with AbsoluteIDQTM p180 kit (BIOCRATES Life Sciences AG). Results: Several metabolites concentrations changed significantly between the two visits in the M2F group including DL-carnitine, octadecanyl-L-carnithine, ornithine, threonine, alpha-amino adipic acid, hydroxyproline, phosphatidylcholine diacyl C34:3 and ornithine/arginine ratio (FDR < 0.05, p < 0.05). In the F2 M group there was only a significant change in ornithine. After adjustment for age, lifestyle factors and body composition in a linear mixed effects model, these results remained significant but vanished after adjusting for sex hormone levels. This indicates a direct effect of sex hormones on metabolite levels. Opposite effects were seen for changes in ornithine levels between the two groups. This is also in line with sex-specific findings from the population based KORA study. Ornithine was further strongly positively related to testosterone levels in both groups. Conclusion: In this first pilot study we could show that cross-sex hormone treatment induces several changes in serum metabolites. Opposite effects were exerted on Ornithine levels indicating a direct effect of sex-hormones on citric acid cycle regulation.