Carotenoids are a class of isoprenoids widely distributed in plants, algae, fungi and bacteria. Carotenoids are essential components for human diet, providing health promoting and nutritional benefits. Fruits are the major source of carotenoids for human consumption. Carotenoid biosynthesis and regulation in fruits are of great importance for development and maintenance of nutritional quality. In recent years, significant progress has been made in understanding the biosynthesis and regulation of carotenoids in tomato and other widely consumed fruits. Carotenoid accumulation in fruits is highly regulated by developmental programs, environmental factors, and metabolic signals at multiple levels. In this review, we highlight recent insights into transcriptional (transcription factor, alternative RNA splicing,epigenetic modification, miRNA), post-transcriptional and hormone regulation of carotenoid biosynthesis in plants, especially in fruits.