RNA silencing plays a crucial role in regulating gene expression through small(s)RNA, governing several processes such as development, stress responses, and maintenance of genomic integrity. Central to this regulatory mechanism is the loading of sRNAs into ARGONAUTE(AGO) proteins. Arabidopsis thaliana encoded ten AGO proteins. Among these, AGO1 serves as the main effector of microRNA(miRNA)-mediated gene silencing. MiRNAs are transcribed as larger RNA precursors, which are subsequently processed by DICER-LIKE1 in the nucleus releasing the mature miRNAs. Interestingly, miRNA-mediated gene regulation occurs in the cytoplasm, coinciding with the steady-state subcellular localization of AGO1. Previous studies have shown that AGO1 can load miRNAs in the nucleus and then translocate AGO1:miRNA complexes to the cytoplasm. However, the mechanism underlying AGO1 nuclear import has remained elusive. Here, we identified a Nuclear Localization Signal(NLS) within AGO1, which interacts with IMPORTINα protein. Mutation of this NLS in AGO1 abolishes its interaction with IMPORTINα, resulting in exclusive cytoplasmic localization. Complementation experiments using nuclear-localized AGO1 partially restored the ago1-27 mutant, whereas cytoplasmic-only AGO1 fully complemented the mutation at both phenotypic and molecular levels. Immunoprecipitation assays confirmed that cytoplasmic-only AGO1 retains miRNA loading capabilities, indicating a cytoplasmic loading mechanism in addition to the previously described nuclear loading. Nonetheless, competition assays between plants expressing at the same time nuclear-only and cytoplasmic-only AGO1 revealed distinct sRNA loading preferences, with nuclear-only AGO1 preferentially loading miRNAs and cytoplasmic-only AGO1 exhibiting quantitative loading of siRNAs. Collectively, these findings validate the existence of a dual cytoplasmic and nuclear sRNA loading activity of AGO1.