In Arabidopsis thaliana, microRNAs (miRNAs) are mainly loaded into ARGONAUTE1 (AGO1) to posttranscriptionally regulate gene expression. We previously found that ENHANCED MiRNA ACTIVITY1 (EMA1), an importin ß family protein, negatively regulates miRNA loading into AGO1. In this study, through a suppressor screening of ema1, we identified another importin ß protein, TRANSPORTIN1 (TRN1), as a regulatory component in the miRNA pathway. Mutation of TRN1 did not reduce miRNA accumulation, but it impaired miRNA activity. We found that TRN1 interacted with AGO1. Mutation of the three conserved residues required for cargo recognition of TRN1 reduced its interaction with AGO1 and compromised its function in regulating miRNA activity. Intriguingly, TRN1 dysfunction did not change the cytoplasmic-nuclear distribution of miRNAs and AGO1 but reduced the amount of miRNAs associated with AGO1. These results indicate that TRN1 positively regulates miRNA activity by promoting the association of miRNAs with AGO1, and they reveal opposing roles of two importin ß family proteins in miRNA loading.