The phenotypic variations that follow polyploidization are expected to improve agricultural productivity and efficiency [1]. However, the effect of polyploidization on plant metabolism has rarely been studied. This study evaluated the metabolic alterations that followed autotetraploidization in the fruit of Ponkan mandarin (C. reticulata Blanco) for three consecutive years and explored the underlying changes to the transcriptome. The autotetraploid (4x) Ponkan fruit had higher levels of total acids, ascorbic acid and total phenolic compounds than the diploid (2x). The primary metabolites especially the organic acids tended to accumulate at higher levels in the 4x fruit. Conversely, two major groups of secondary metabolites (i.e. flavonoids and carotenoids) tended to accumulate at lower levels. The expression levels of citric acid biosynthesis-related genes were unaltered in 4x fruit compared to the 2x fruit. Additionally, genes associated with the transport and utilization of citric acid were significantly down-regulated during ripening, which might induce increases in the levels of citric acid in the 4x fruit. Lower levels of flavonoids and carotenoids in the 4x fruit are potentially associated with decreases in the transport and utilization of citric acid, which is an important metabolite. Citric acid contributes to respiration by serving as an intermediated in the tricarboxylic acid cycle (TCA) and also provides carbon for the production of secondary metabolites. This study demonstrates that polyploidization can influence metabolism in plants.