Pectin is an important structural part of main cell walls of plant whereby pectinases are a large group of enzymes that break down pectin into simpler molecules. Pectinases are present in the fruits of plants where they play a natural role in the ripening process. They are categorized into esterases and depolymerase on the basis of pectinase’s mode of action on pectin. This enzyme is important in the biotechnological sector with gradual increase in their market. They hold a prominent position among the commercially produced industrial enzymes. Fungal base pectinases are applied for large-scale production because they are easy to maintain and multiply under controlled environments. Pectinases produced from fungal sources have low activity and are less tolerant of alkaline, acidic, and high-temperature conditions. These enzymes are eco-friendly tools of nature that are being used widely in various industrial sectors. In industrial sector, pectinases are mainly involved in wine and paper manufacturing industry. In food sector, these enzymes are utilized for extraction, clarification, and stabilization of fruit juices. They also improve juice yield and involved in cocoa, coffee, and tea fermentation. This chapter describes the pectinolytic enzymes and their substrates, the fungal pectinase production and characterization, and the industrial application of these enzymes.