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The Crested Ibis (Nipponia nippon) has long fascinated ornithologists with its enigmatic plumage color change. After more than a century of curiosity, the mystery was finally unraveled in the 1970s, unveiling the mechanism behind this remarkable transformation. Unlike other bird species, the Crested Ibis achieves its nuptial plumage coloration through a unique daubing behavior. After a water-bathing, it applies a sticky black substance secreted by a patch of skin in the neck and throat region. However, the chemical components of this black substance have not been studied in detail until now. To address this issue, we conducted a study to detect the components of the black substance and explore the relationship between sex hormone levels and the secretion of the black substance. We used enzyme-linked immunosorbent assay (ELISA) to measure the monthly changes in steroid hormone levels (estradiol E2, testosterone T, and progesterone PROG) levels in feces. We also analyzed the correlation between sex hormone levels and daubing behavior. The results showed that the sex hormone levels are closely related to the secretion and application of the black substance. In addition, we qualitatively analyzed the chemical components of the black substance using gas chromatography–mass spectrometry (GC-MS), uncovering the presence of 117 distinct chemical components. We assume that the black coloration results from the polymerization of selected chemical constituents among these components. These findings provide a groundwork for further exploration into the biological significance of the black substance. Overall, our study detected components in the black substance and studied how sex hormone levels relate to its secretion. Understanding the hormone effects on coloration helps in precise habitat management, like wetland preservation, crucial for Crested Ibis survival. Implementing hormone-boosting measures during breeding seasons enhances reproduction and health, vital for their conservation. Full article