MicroRNAs (miRNAs), are similar to 22 nucleotides long, non-coding RNAs that control gene expression post-transcriptionally by binding to their target mRNA's 3'UTRs (untranslated regions). Due to their roles in various important regulatory processes and pathways, miRNAs have been implicated in disease mechanisms such as tumorigenesis when their expression is deregulated. To date, a significant number of miRNAs and their target messenger RNAs (mRNAs) have been identified and verified. It is generally accepted that miRNAs can potentially bind to many mRNAs, which brings the requirement of validation of these interactions. While understanding that such individual interactions is crucial to delineate the role of a specific miRNA, we took a holistic approach and analyzed global changes in the cell due to expression of a miRNA in a model cell line system. Our model consisted of MCF7 cells stably transfected with miR-125b (MCF7-125b) and empty vector (MCF7-EV). MiR-125b is one of the known down-regulated miRNAs in breast cancers. In this study we examined the global structural changes in MCF7 cells lacking and expressing miR-125b by Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy and investigated the dynamic changes by more sensitive spin-labelling Electron Spin Resonance (ESR) spectroscopy. Our results revealed less RNA, protein, lipid, and glycogen content in MCF7-125b compared to MCF7-EV cells. Membrane fluidity and proliferation rate were shown to be lower in MCF7-125b cells. Based on these changes, MCF7-125b and MCF7-EV cells were discriminated successfully by cluster analysis. Here, we provide a novel means to understand the global effects of miRNAs in cells. Potential applications of this approach are not only limited to research purposes. Such a strategy is also promising to pioneer the development of future diagnostic tools for deregulated miRNA expression in patient samples.