Background <p>Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder associated with impaired bile flow and increased perinatal risks. Xenin-25, a gastrointestinal peptide regulating bile flow and metabolic activity, has not been investigated in ICP. This study aimed to evaluate serum Xenin-25 levels in ICP and to assess its diagnostic and prognostic potential.</p> Methods <p>The present prospective cross-sectional study comprised 44 women with ICP and 44 healthy controls of equivalent gestational age. The diagnosis of ICP was established on the presence of pruritus and fasting bile acid levels that exceeded 10 µmol/L. Serum concentrations of Xenin-25 were measured by means of the enzyme-linked immunosorbent assay (ELISA). A comprehensive data set encompassing demographic and clinical characteristics was systematically collected. Group comparisons were performed using a range of analytical methods, including t-tests, Mann–Whitney U tests and chi-square tests. Correlations were analysed to provide a comprehensive overview of the data. The diagnostic accuracy was determined by receiver operating characteristic (ROC) analysis.</p> Results <p>Levels of Xenin-25 were found to be significantly elevated in ICP cases in comparison to the control group (48.3 ± 7.9 vs. 20.9 ± 5.2; <i>p</i> &lt; 0.001). ROC analysis yielded an area under the curve (AUC) value of 1.000, indicating perfect sensitivity and specificity of 100% and 97.7%, respectively. The correlation between Xenin-25 and various biological markers was investigated, with a strong correlation observed between Xenin-25 and alanine aminotransferase ALT (<i>r</i> = 0.549), aspartate aminotransferase AST (<i>r</i> = 0.473), aspartate aminotransferase to platelet ratio index APRI (<i>r</i> = 0.445), fibrinogen (<i>r</i> = 0.610), and Fib-4 (<i>r</i> = 0.351). However, a negative correlation was identified between Xenin-25 and albumin (<i>r</i>=-0.305, <i>p</i> = 0.004). No significant correlation was observed with maternal age, gestational week, or bile acids. In comparison with the control group, the ICP group exhibited lower gestational age at delivery and reduced birth weight (<i>p</i> &lt; 0.001). Furthermore, there were elevated rates of adverse outcomes, although no direct correlation was observed between Xenin-25 and perinatal outcomes.</p> Conclusions <p>This initial investigation into Xenin-25 in ICP demonstrates its substantial elevation and exceptional diagnostic efficacy, eclipsing conventional markers. The findings, indicating a link between Xenin-25, hepatic stress and fibrosis scores, support the hypothesis that Xenin-25 can function as a prognostic biomarker. In order to confirm its role in intrahepatic cholestasis of pregnancy (ICP) pathophysiology and clinical practice, larger multicentre studies are required.</p> Trial registration <p>Not applicable.</p>