Previous studies have proven that short-day treatment is an effective means to regulate shoot growth and abiotic stress resistance in conifers. However, the effects on Chinese pine (Pinus tabuliformis) seedlings are less well known. In this study, short-day treatments were applied to container-grown Chinese pine seedlings by artificially reducing day length to 8 or 10h for 3weeks. Growth and physiological responses of the seedlings were evaluated. In addition, proteome analysis using two-dimensional electrophoresis in combination with MALDI-TOF/TOF MS analysis was performed on needles of the seedlings. Short-day treatments significantly affected seedling bud set, height, root collar diameter and number of first-order lateral roots (>1mm diameter at junction with taproot). Short-day treatments significantly enhanced the content of total chlorophyll, starch, abscisic acid, and lignin, and increased activity of phenylalanine ammonialyase, polyphenol oxidase, and peroxidase, but did not differ in the content of soluble sugar. We positively identified eight differentially expressed proteins induced by short-day treatments: oxygen-evolving enhancer protein 1, transcription factor VOZ1, ferredoxin-NADP reductase, leaf isozyme 1, protein MET1, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase 1, elongation factor Tu, and ribulose bisphosphate carboxylase/oxygenase activase. These proteins are mainly involved in photosynthesis, carbohydrate metabolism, as well as abiotic and biotic stress resistance. Our results suggest that short-day treatment is a way to produce high-quality Chinese pine seedlings in the nursery.