Emerging only around the turn of the millennium, microRNAs (miRNAs) mediating post-transcriptional gene silencing have been rapidly recognized as central regulators of eukaryotic gene expression, affecting all aspects of multi-cellular life and disease. Intracellular pathways such as RNA silencing are often spatially organized, in that biomolecules localize within and/or are transported to distinct sub-cellular granules to mediate specific functions. As one example, miRNA bound messenger RNAs (mRNAs) localize to processing bodies (PBs), sub-cellular foci enriched in RNA processing and degrading enzymes, as either a cause or consequence of gene silencing processes critical for cancer suppression. To gain a deeper understanding of the hitherto poorly understood nature of miRNA:PB interactions, to more accurately quantify the distribution of miRNAs within PBs, and to unravel the relative contribution of PBs in miRNA-mediated gene silencing, we integrated our recently developed iSHiRLoC1,2,3 (intracellular Single molecule High-Resolution Localization and Counting) technique with systems biology modeling. For intracellular experiments, miRNAs and PBs were labeled with spectrally distinct fluorescent probes so that single particles of microinjected and functionally fluorophore labeled miRNAs could be co-localized and co-tracked with individual PBs inside human cells with ~30 nm/50 ms spatiotemporal resolution. We found that only a small fraction (~10%) of miRNAs resides within PBs, predominantly as monomeric or dimeric micro-ribonucleoprotein complexes (miRNPs). Strikingly, the residence time of miRNAs within PBs varies from a few hundred milliseconds to many seconds, with individual particles docking with very distinct (heterogeneous) residence times. Moreover, both the residence time and extent of heterogeneity are dependent on the gene regulatory potential of a miRNA: let-7 miRNA with >800 distinct mRNA targets resides in PBs on average ~1 s through both stable and transient associations, whereas a CXCR4 miRNA with <80 mRNA targets generally docks to PBs stably with an average dwell time of ~3 s. Taken together, our experimental data reveal that a multiplicity of targets further weakens sparse miRNA:PB interactions, suggesting a relatively minor role of PBs in gene silencing. Accordingly, using our experimental iSHiRLoC data as input in Monte-Carlo based on-lattice kinetic modeling predicted that a large number of small, dispersed, highly mobile PBs should be more effective in miRNA-mediated mRNA degradation than the typically observed fewer large, immobile PBs. Varying the ionic strength of the culturing medium as an efficient modulator of intracellular PB dispersity, we experimentally tested and confirmed this prediction. Our single molecule systems biology approach opens up an avenue toward better understanding the mechanistic underpinnings of RNA silencing pathways in healthy and cancer-diseased cells.