Nesprins are highly conserved spectrin-repeat containing scaffold proteins pre-dominantly known to function at the nuclear envelope (NE). However, nesprin isoforms are emerging with localizations and scaffolding functions at sites away from the NE, suggesting their functions are more diverse than originally thought. In this study, we combined nesprin-1 coimmunoprecipitations with mass spectrometry to identify novel nesprin-1 binding partners for isoforms that localize to subcellular compartments beyond the NE. We show that one of these interactors, matrin-3, localizes to mRNA Processing bodies (PBs), where we have previously shown a nesprin-1 isoform to localize. Furthermore, we show that matrin-3 is part of PB mRNP complexes, is a regulator of miRNA-mediated gene silencing and possibly shuttles to stress granules in stressed cells. More importantly, we identify a new C-terminally truncated matrin-3 isoform which is likely to be involved in these functions and PB localization. This study highlights several novel nesprin-1 binding partners as well as a new function and localization for matrin-3 in cytoplasmic RNA granules.