Objective: This study aimed to elucidate the regulatory mechanism of an auxin amide hydrolase gene (IAA-Leucine Resistant1-like Hydrolase, RhILL1) in the petal pigmentation of rose (Rosa hybrida), providing theoretical insight into the hormonal regulation of flower coloration at the molecular level. Methods: Using petals at Stage 3 (S3) of the cut rose cultivar ‘Pink Floyd’ as experimental material, we cloned the rose auxin amide hydrolase gene RhILL1 and validated its function via virus-induced gene silencing (VIGS). The expression levels of anthocyanin biosynthetic genes, anthocyanin content, and auxin (IAA) levels were analyzed to assess the role of RhILL1 in petal pigmentation. Results: The full-length open reading frame (ORF) of RhILL1 was cloned, spanning 1326 bp and encoding a 441-amino-acid protein harboring two conserved domains, Peptidase_M20 and M20_dimer, characteristic of the ILL1 protein family. Functional characterization was performed using VIGS. Quantitative real-time PCR (qRT-PCR) revealed that RhILL1 expression progressively increased from the Green (G) stage to S3, correlating with intensified petal coloration. Silencing RhILL1 resulted in visibly lighter petals, the reduced expression of anthocyanin biosynthetic genes, and a significant decrease in endogenous indole-3-acetic acid (IAA) levels compared with controls. Moreover, exogenous application of 10 μM naphthaleneacetic acid (NAA) to petals significantly preserved petal pigmentation. Conclusion: These findings suggest that RhILL1 contributes to the development and maintenance of petal coloration in rose, likely by modulating IAA levels, thereby influencing the expression of anthocyanin biosynthesis-related genes.