The hypothalamus, particularly its ventromedial and lateral regions, plays a pivotal role in homeostatic appetite regulation and is therefore a significant brain structure in the development of obesity. Additionally, the development of obesity can be caused by improper hedonic regulation, which involves neural circuits and systems associated with pleasure and reward. Several studies indicate a possible link between rubidium (Rb) and obesity, despite this element is not being typically considered influential in vital life processes. The present study, therefore, aims to investigate whether excessive body fat in obese animals alters rubidium levels in brain regions directly or indirectly involved in appetite regulation. The research was conducted on high-calorie diet (HCD)-induced obese rats (OB, n = 8) and their lean counterparts (L, n = 8). The determination of Rb levels in brain areas was performed using synchrotron radiation-based X-ray fluorescence microanalysis (SRXRF). The obtained results show a significantly higher level of Rb in all brain areas examined, although the increase in this element in obese individuals was not the same in all structures. The largest relative difference (over 70%) was observed for the orbitofrontal cortex, and the smallest (about 35%) for the amygdala. Principal component analysis with linear projections demonstrated a clear differentiation between the brain structures of obese and non-obese individuals based on the full elemental composition of tissues, while Rb was the only element that distinguished the obese group in each of the examined brain structures. The results obtained clearly confirm the increase in Rb levels in the brain structures responsible for regulating appetite in obesity.