RNA-binding proteins (RBPs) play critical roles in the regulation of gene expression. Recent studies have begun to detail the RNA recognition mechanisms of diverse RBPs. However, given the array of RBPs studied so far, it is implausible to experimentally profile RBP-binding peaks for hundreds of RBPs in multiple non-model organisms. Here, we introduce MuSIC (Multi-Species RBP–RNA Interactions using Conservation), a deep learning-based framework for predicting cross-species RBP–RNA interactions by leveraging label smoothing and evolutionary conservation of RBPs across 11 phylogenetically diverse species ranging from human to yeast. MuSIC outperforms state-of-the-art computational methods, and achieves highly accurate prediction of RBP-binding peaks across species. The prediction confidence is higher in the metazoan species, partially reflecting differences in RBP conservation patterns. Finally, the effects of homologous genetic variants on RBP binding can be computationally quantified across species, followed by experimental validations. The target transcripts with disrupted binding events are enriched in the ubiquitination-associated pathways. To summarize, MuSIC provides a useful computational framework for predicting RBP–RNA interactions cross-species and quantifying the effects of genetic variants on RBP binding, offering insights into the RBP-mediated regulatory mechanisms implicated in human diseases.