The sugar transporter SWEET (sugars will eventually be exported transporter) family has sugar transportation activity for glucose, fructose or sucrose, which is involved in the phloem loading and unloading of photosynthetic product, as well as physiological processes such as flower, fruit and seed development in higher plant. To explore the physiological functions of CitSWEET15 (Cs7g02970) during fruit development and ripening of sweet orange, gene cloning, expression patterns, subcellular localization and sucrose transport activity were performed in the study. The ORF (open reading frame) of CitSWEET15 gene was 918 bp, which encoded 305 amino acids, and possessed seven transmembrane domains. The theoretical isoelectric point of CitSWEET15 was 6.29, and the relative molecular weight was 34.15 kDa. Phylogenetic analysis suggested that CitSWEET15 together with AtSWEET15, VvSWEET15, LcSWEET15 and PuSWEET15 were orthologous genes, and belonged to the SWEET III clade. Sequence alignment showed that nucleotide and amino acid sequence identities of CitSWEET15 with those of Clementine mandarin, Mangshan wild mandarin, Kumquat, Pomelo, Citrus ichangensis Swingle and Citron were 98.69%-99.9% and 97.38%-100%, respectively. CitSWEET15 and the mentioned Citrus SWEET15 members were completely consistent with four sucrose recognition sites and three sucrose transport activity sites of AtSWEET13, which suggested that all CitSWEET15 and Citrus SWEET15 members may have sucrose transport activity. Quantitative real-time PCR analysis demonstrated that the expression of CitSWEET15 was high in the seed and fruit pulp of sweet orange, the expression level of CitSWEET15 in fruit pulp was low at 80-140 days after flowering, then significantly up-regulated as fruit development, and reached the highest at 230 days after flowering. CitSWEET15 was fused with green fluorescent protein at the C-terminus, and then transiently expressed in Arabidopsis mesophyll protoplasts. The subcellular localization analysis suggested the fluorescence signal was distributed around the outer edge of protoplast, indicating that CitSWEET15 was located in the plasma membrane. By heterologous expression in Saccharomyces cerevisiae W303a (W303-1A) strain and incubation in the sucrose analogue Esculin, the fluorescence intensity was detected to test the sucrose transportation activity of CitSWEET15 and AtSWEET10 (At5g50790, positive control). The fluorescent intensity of yeast cells expressing CitSWEET15, as similar as AtSWEET10, was significantly higher than vector control, indicating that CitSWEET15 had sucrose transportation activity. In conclusion, CitSWEET15 is highly conserved in Citrus species, functions as a plasma membrane sucrose transporter, and involves in apoplastic sucrose transport during fruit ripening of sweet orange. The results would provide a basis for understanding the soluble sugar accumulation in sweet orange fruit, and a candidate gene for improving fruit flavor and quality.