Iron-sulfur [Fe-S] clusters, acting as co-factors for iron-sulfur proteins, are ubiquitously implicated in a diverse array of biological processes, such as photosynthesis, respiration, electron transport, and the biosynthesis of essential vitamins and co-factors. Their intracellular biogenesis is modulated by a suite of proteins that catalyze and regulate the process, with compartmentalization occurring within discrete subcellular compartments. Mitochondria, as the central organelles for cellular energy metabolism, harbor numerous key metabolic enzymes that are iron-sulfur proteins, necessitating the provision of Iron-sulfur clusters by the mitochondrial assembly machinery, ISC (Iron-sulfur cluster). Advancements in research, particularly from bacteria and yeast systems, have facilitated significant strides in the identification and functional characterization of pivotal catalytic and regulatory proteins within the plant mitochondrial ISC system. Additionally, research into the role of Iron-sulfur clusters in the growth and development of plants has made considerable progress. The elucidation of plant-specific components within the Iron-sulfur cluster synthesis machinery and the mechanisms by which this system responds to environmental stress are areas of growing interest. This manuscript provides a comprehensive review of the current state of research on the mechanism of Iron-sulfur cluster synthesis in plants, with a particular focus on the mitochondrial ISC assembly system. It also offers a succinct synopsis of the role of key genes within the ISC system in plant growth and development, as well as their involvement in the response to abiotic stressors.