Background: FIRE-3 compared first-line therapy with FOLFIRI plus cetuximab (cet) or bevacizumab (bev) in KRAS exon 2 wild-type (wt) patients with metastatic colorectal cancer. Recent analyses showed mircoRNA-21 (miR-21) expression level may be a predictive biomarker for anti-EGFR-therapy raising the question whether miR-21 influences gene expression in the EGFR signaling pathway. Methods: Reverse-transcription quantitative polymerase chain reaction assay identified quantitative miR-21 expression. Median expression was defined as a threshold value to discriminate FIRE-3 population into miR-21 low and high groups. Differential gene expression based on additional mRNA microarray data (Almac Inc, Xcel Array) was calculated by linear models adjusted for multiple testing followed by single sample gene set enrichment analysis (ssGSEA) to compare differentially enriched hallmarks of cancer gene sets. Overall response rate (ORR) was compared using Fisher´s exact test. Median progression-free (PFS) and overall survival (OS) were analyzed using Kaplan-Meier estimation and log-rank test. Results: 333 RAS wt patients provided material for miR-21 expression analysis. In these patients, low miR-21 expression was associated with higher ORR (80.0% vs. 57.9%; p = 0.005) and longer OS (35.8 months (mo) vs. 25.9 mo; p = 0.005) when cet vs bev was added to FOLFIRI. High miR-21 expression was associated with comparable ORR (74.6% vs. 64.0%; p = 0.21) and OS (24.5 mo vs. 23.8 mo; p = 0.4). There was no significant difference in PFS in either group. By comparing miR-21 low and high groups using normalized mRNA microarray data, 538 genes were found to be significantly differentially expressed in RAS wt patients after adjustment for multiple testing. Including data from the two groups into ssGSEA yielded 23 hallmark of cancer gene sets that were significantly differentially enriched; among them, KRAS-signaling showed higher enrichment in the miR-21 high group (adjusted p = 2.09 E-13). Conclusions: MiR-21 expression level might be a predictive biomarker for anti-EGFR-therapy by modulating KRAS signaling in FIRE-3 patients.