This work reports the development of suspension culture system of transgenic Marsh grapefruit (Citrus paradisi Macf., Rutaceae) callus overexpressing bacterial phytoene synthase and the use of this suspension culture to investigate the effects of β-cyclocitral on carotenoid content and composition. At a β-cyclocitral concentration of 0.5 mM and after ten days cultivation, analysis of the carotenoids showed a significant increase in the content of β-, α-carotene, and phytoene predominantly. The maximal increase in total provitamin A carotenoids content following β-cyclocitral application was ∼2-fold higher than the control, reaching 245.8 μg/g DW. The trend for increased transcript levels of biosynthetic genes PSY and ZDS correlated with the enhancement of the content of these carotenes following β-cyclocitral treatment and GC-MS based metabolite profiling showed significant changes of metabolite levels across intermediary metabolism. These findings suggest that β-cyclocitral can act as a chemical elicitor, to enhance the formation of carotenes in citrus suspension-cultured cells (SCC), which could be utilized in studying the regulation of carotenoid biosynthesis and biotechnological application to the renewable production of nutritional carotenoids.