Neuropeptides are involved in numerous brain activities being able to control a wide spectrum of physiological functions. In recent years, a number of novel pleiotropic regulatory peptides have been discovered in animal brain structures. The purpose of this descriptive neurochemical investigation was to detect the possible expression of the novel multifunctional neuropeptides spexin (SPX) and nesfatin-1 within the human brainstem. Using immunohistochemical and fluorescence techniques, neuroanatomical analysis of the SPX and nesfatin-1 expression and distribution was performed in selected sections of the human midbrain and medulla oblongata. The presence of SPX-positive neurons in the human brainstem was revealed for the first time and previous reports on the expression of nesfatin-1 were additionally confirmed. The research results suggest that SPX and nesfatin-1 are new regulatory neuropeptides of the human brainstem potentially involved in the regulation of key autonomic activities of this brain region.