The Pistacia chinensis Bunge is traditionally dioecious, and the female trees are more required to grow in practice for oil seed production. The discovery of monoecious P. chinensis Bunge in North China provided good raw materials to study the sex differentiation process. The objective of this study was to identify the differently expressed proteins in flower buds in two key sex differentiation phases in monoecious P. chinensis Bunge. Morphological observation and paraffin section were used to determine the key phenophases, and label-free quantitative technique was used for proteomic analysis. The results showed that the proteins related to oxidative stress resistance up-regulated while proteins involved in photosynthesis down-regulated during the female primordium differentiation in bisexual flower buds of the monoecious P. chinensis Bunge in early March, while proteins related to oxidative stress resistance, ribosome activity, and photosynthetic function up-regulated during the male primordium differentiation in bisexual flower buds of the monoecious P. chinensis Bunge in late May. The most up-regulated proteins all involved in the photosynthesis pathway in both kind of flower buds in late May compared to those in early March, and the down-regulated proteins all involved in the ribosome pathway. The identified differentially expressed proteins such as the Cu/Zn superoxide dismutases may be possible molecular markers for sex determination in monoecious P. chinensis Bunge.