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Abstract Background Nesfatin-1 is a neuropeptide that regulates the hypothalamic-pituitary-gonadal axis and may play a role in uterus function. It is co-expressed with other peptides, such as phoenixin, which can influence sex hormone secretion. Our previous research has confirmed that phoenixin-14 is involved in the development of cystic endometrial hyperplasia (CEH) and pyometra in dogs. Therefore, based on the similarities and interactions between these neuropeptides, we hypothesized that nesfatin-1 might also regulate the reproductive system in dogs. This study aimed to determine the expression of nesfatin-1 and its interaction with phoenixin-14 in dogs with CEH or pyometra compared to healthy females, and concerning animals’ body condition score (BCS 4–5/9 vs. BCS > 5/9). Results The analysis of nesfatin-1 in the uterus of bitches consisted of qPCR, western blot and immunofluorescence assays, and ELISAs. The results showed significantly higher nesfatin-1 encoding gene, nucleobindin-2 mRNA (Nucb2) and nesfatin-1 protein expression in overweight females and those suffering from CEH or pyometra compared to healthy animals. The immunoreactivity of nesfatin-1 was elevated in the uteri of bitches with higher BCS > 5/9. Moreover, nesfatin-1 blood concentrations increased in all examined overweight bitches. In the case of phoenixin signals, we found opposite results, regardless of the female body condition score. Conclusion The etiology of CEH and pyometra are not fully known, although we have expanded the level of knowledge with respect to the possible interaction of nesfatin-1 and phoenixin in female dogs’ uteri. They interact oppositely. With increasing female body weight, the expression of nesfatin-1 in the uterus and its peripheral blood concentration increased. However, for female dogs affected by CEH and pyometra, a decreased level of phoenixin-14, irrespective of their body condition score is characteristic. This knowledge could be crucial in the development of biomarkers for these conditions, which may lead to earlier recognition.