Interstock is an important agronomic technique for regulating plant growth and fruit quality, and overcoming the incompatibility between rootstocks and scions; however, the underlying mechanisms remain largely unknown. In this study, the effects and regulatory mechanisms of tangor grafting, with and without interstocks, on the growth and development of scions were analyzed by combining morphology, physiology, anatomy and transcriptomics. Morphological and physiological analyses showed that interstocks (‘Aiyuan 38’ and ‘Daya’) significantly improved the growth of seedlings, effectively enhanced the foliar accumulation of chlorophyll and carotenoids, and increased the thickness of leaf tissues. Using ‘Aiyuan 38’ as the interstock, photosynthetic efficiency and starch content of citrus seedlings improved. Transcriptomics showed that genes related to photosynthesis and photosynthetic antenna proteins were upregulated in interstock-treated seedlings, with significant upregulation of photosystem PSI- and PSII-related genes. In addition, multiple key genes may be involved in plant hormone signaling, starch and sucrose metabolism, and transcriptional regulation. Taken together, these findings provide novel insights into the role of interstocks in regulating and contributing to the growth and development of grafted seedlings, and will further define and deploy candidate genes to explore the mechanisms of rootstock-interstock-scion interactions.