Several miRNAs are conserved in different plant families, but their abundance and target genes vary between species, organs, and stages of development. Target genes of miRNAs are mostly transcription factors, involved in the control of many plant developmental processes, including fruit development. MiR164 is a conserved miRNA, highly expressed in fruits, and is validated to target a subset of genes of the NAC-domain transcription factor gene family. The objective of this work was to analyze the phenotypic effects of the constitutive expression of miR164 during the life cycle of Arabidopsis and tomato. MiR164 overexpression (164-OE) lines for Arabidopsis and tomato were generated and analyzed during plant development. The constitutive miR164 expression showed that miR164 affected the morphology of Arabidopsis and tomato, and it affected the transition from the vegetative to the reproductive phase in Arabidopsis. Moreover, the miR164 overexpression affected the time required for each developmental stage of tomato fruit. These results suggest that miR164 plays general and specific roles during development in Arabidopsis and tomato, including fruit development, which could be exploited for the improvement of traits of agronomic interest in important species.