Ewing sarcoma (EwS) is an aggressive cancer characterized by chromosomal translocations generating fusions of the EWSR1 gene with ETS transcription factors (in 85% FLI1). EWSR1-FLI1 induces gene expression via binding to enhancer-like GGAA-microsatellites, whose activity correlates with the number of consecutive GGAA-repeats. Herein we investigate the role of the secretory neuropeptide CALCB (calcitonin-related polypeptide β) in EwS, which signals via the CGRP (calcitonin gene-related peptide) receptor complex, containing RAMP1 (receptor activity modifying protein 1) as crucial part for receptor specificity. Analysis of 2678 gene expression microarrays comprising 50 tumor entities and 71 normal tissue types revealed that CALCB is specifically and highly overexpressed in EwS. Time-course knockdown experiments showed that CALCB expression is tightly linked to that of EWSR1-FLI1. Consistently, gene set enrichment analyses of genes whose expression in primary EwS is correlated to that of CALCB indicated that it is co-expressed with other EWSR1-FLI1 target genes and associated with signatures involved in stemness and proliferation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for FLI1 and histone marks from EwS cell lines demonstrated that EWSR1-FLI1 binds to a GGAA-microsatellite close to CALCB, which exhibits characteristics of an active enhancer. Reporter assays confirmed the strong EWSR1-FLI1- and length-dependent enhancer activity of this GGAA-microsatellite. Mass spectrometric analyses of EwS cell culture supernatants demonstrated that CALCB is secreted by EwS cells. While short-term RNA interference-mediated CALCB knockdown had no effect on proliferation and clonogenic growth of EwS cells in vitro, its long-term knockdown decreased EwS growth in vitro and in vivo. Similarly, knockdown of RAMP1 reduced clonogenic/spheroidal growth and tumorigenicity, and small-molecule inhibitors directed against the RAMP1-comprising CGRP receptor reduced growth of EwS. Collectively, our findings suggest that CALCB is a direct EWSR1-FLI1 target and that targeting the CALCB/RAMP1 axis may offer a new therapeutic strategy for inhibition of EwS growth.