Hemolymph is vital for the flow and transportation of nutrients, ions, and hormones in the honey bee and plays role in innate immune defense. The proteome of the hemolymph changes over the life of a honey bee, but many of these changes are not well characterized, including changes during the life cycle transition from the larval to pupal stages of workers. We used two-dimensional gel electrophoresis, mass spectrometry, bioinformatics, and Western blot to analyze the proteome changes of the honeybee hemolymph during the transition from newly hatched larvae to five-day-old pupae. Of the 49 nonredundant proteins that changed in abundance (identified by 80 protein spots), 29 (59.2%) and 20 (40.8%) were strongly expressed in the larvae and the pupae, respectively. The larval hemolymph had high expressions of major royal jelly proteins and proteins related to metabolism of carbohydrates and energy, folding activities, development, and the cytoskeleton and antioxidant systems. Proteins involved in food storage and the metabolism of fatty acids and amino acids were abundantly expressed during the late larval to pupal development stages. The proteins expressed by the young larvae are used to enhance their development process and as a temporal innate immune protection mechanism until they gain immunity with age development. The pupae use more energy storage related proteins as they prepare for their non-diet-driven pupation. Our data provide new evidence that changes in the hemolymph at the proteome level match the processes during life transitions in the honeybee.