The yellow-fleshed loquat is abundant in carotenoids, which determine the fruit’s color, provide vitamin A, and offer anti-inflammatory and anti-cancer health benefits. In this research, the impact of abscisic acid (ABA), a plant hormone, on carotenoid metabolism and flesh pigmentation in ripening loquat fruits was determined. Results revealed that ABA treatment enhanced the overall content of carotenoids in loquat fruit, including major components like β-cryptoxanthin, lutein, and β-carotene, linked to the upregulation of most genes in the carotenoid biosynthesis pathway. Furthermore, a transcription factor, EjWRKY6, whose expression was induced by ABA, was identified and was thought to play a role in ABA-induced carotenoid acceleration. Transient overexpression of EjWRKY6 in Nicotiana benthamiana and stable genetic transformation in Nicotiana tabacum with EjWRKY6 indicated that both carotenoid production and genes related to carotenoid biosynthesis could be upregulated in transgenic plants. A dual-luciferase assay proposed a probable transcriptional control between EjWRKY6 and promoters of genes associated with carotenoid production. To sum up, pre-harvest ABA application could lead to carotenoid biosynthesis in loquat fruit through the EjWRKY6-induced carotenoid biosynthesis pathway.