MicroRNA408 (miR408) is an ancient and highly conserved miRNA, which is involved in the regulation of plant growth, development and stress response. However, previous research results on the evolution and functional roles of miR408 and its targets are relatively scattered, and there is a lack of a systematic comparison and comprehensive summary of the detailed evolutionary pathways and regulatory mechanisms of miR408 and its targets in plants. Here, we analyzed the evolutionary pathway of miR408 in plants, and summarized the functions of miR408 and its targets in regulating plant growth and development and plant responses to various abiotic and biotic stresses. The evolutionary analysis shows that miR408 is an ancient and highly conserved microRNA, which is widely distributed in different plants. miR408 regulates the growth and development of different plants by down-regulating its targets, encoding blue copper (Cu) proteins, and by transporting Cu to plastocyanin (PC), which affects photosynthesis and ultimately promotes grain yield. In addition, miR408 improves tolerance to stress by down-regulating target genes and enhancing cellular antioxidants, thereby increasing the antioxidant capacity of plants. This review expands and promotes an in-depth understanding of the evolutionary and regulatory roles of miR408 and its targets in plants.