MicroRNAs (miRNAs) are an important class of post-transcriptional regulators of gene expression that are involved in various cellular and phenotypic processes. A number of studies have shown that miRNA expression is induced by signaling pathways. Moreover, miRNAs emerge as regulators of signaling pathways. Here, we present the miTALOS web resource, which provides insight into miRNA-mediated regulation of signaling pathways. As a novel feature, miTALOS considers the tissue-specific expression signatures of miRNAs and target transcripts to improve the analysis of miRNA regulation in biological pathways. MiTALOS identifies potential pathway regulation by (i) an enrichment analysis of miRNA targets genes and (ii) by using a proximity score to evaluate the functional role of miRNAs in biological pathways by their network proximity. Moreover, miTALOS integrates five different miRNA target prediction tools and two different signaling pathway resources (KEGG and NCI). A graphical visualization of miRNA targets in both KEGG and NCI PID signaling pathways is provided to illustrate their respective pathway context. We perform a functional analysis on prostate cancer-related miRNAs and are able to infer a model of miRNA-mediated regulation on tumor proliferation, mobility and anti-apoptotic behavior. miTALOS provides novel features that accomplish a substantial support to systematically infer regulation of signaling pathways mediated by miRNAs.