The expression of microRNAs is altered in various cancer types, leading to their definition as onco- and tumor-suppressor microRNAs. In our study, we investigated the role of miR-335 in the formation of sporadic human breast cancer and its involvement in the regulatory network of the breast cancer susceptibility geneBRCA1. To validate single components of theBRCA1cascade, microRNA overexpression was performed in a cell culture model with subsequent protein analysis and luciferase reporter assays. Here, we were able to identify miR-335 as simultaneously regulating the knownBRCA1activators ERα, IGF1R, SP1 and the repressor ID4, including a feedback regulation of miR-335 expression by estrogens. Overexpression of miR-335 resulted in an upregulation ofBRCA1mRNA expression, suggesting a functional dominance of ID4 signaling. The relevance of the miR-335 regulation for human breast cancer was confirmed in primary sporadic breast cancer specimens with significantly decreased miR-335 levels (p< 0.05) in comparison to normal controls. Interestingly, the microRNA expression level correlated positively to theBRCA1transcript level, supporting the hypothesis of a miR-335-mediated regulation of the tumor suppressor gene. Functionally, overexpression of miR-335 led to decreased cell viability and an increase in apoptosis, supporting its tumor-suppressive function. In summary, our data indicate that miR-335 affects different targets in the upstreamBRCA1-regulatory cascade with impact on key cellular functions such as proliferation and apoptosis. Deregulation of the microRNA during breast cancer development and progression may thereby lead to an increased tumorigenic potential by inactivating crucial tumor-suppressive signals.