Background: The growth factor heregulin (HRG) potently stimulates epithelial cell survival and proliferation through the binding of its cognate receptor ErbB3 (also known as HER3). ErbB3-dependent signal transmission relies on the dimerization partner ErbB2, a receptor tyrosine kinase that is frequently overexpressed and/or amplified in breast cancer cells. Substantial evidence suggests that deregulated ErbB3 expression also contributes to the transformed phenotype of breast cancer cells. Results: By genome-wide screening, we identify 43 microRNAs (miRNAs) that specifically impact HRG-induced activation of the PI3K-Akt pathway. Bioinformatic analysis combined with experimental validation reveals a highly connected molecular miRNA-gene interaction network particularly for the negative screen hits. For selected miRNAs, namely miR-149, miR-148b, miR-326, and miR-520a-3p, we demonstrate the simultaneous downregulation of the ErbB3 receptor and multiple downstream signaling molecules, explaining their efficient dampening of HRG responses and ascribing to these miRNAs potential context-dependent tumor suppressive functions. Conclusions: Given the contribution of HRG signaling and the PI3K-Akt pathway in particular to tumorigenesis, this study not only provides mechanistic insight into the function of miRNAs but also has implications for future clinical applications.