Flooding and drought are abiotic stresses that inhibit the growth and yield of soybean. To determine the mechanism involved in response of soybean to flooding and drought stresses, endoplasmic reticulum proteomics was performed. Endoplasmic reticulum fractions were purified from root tips of soybeans and purity was analyzed with enzyme assay, indicating that endoplasmic reticulum was significantly enriched. Proteins of endoplasmic reticulum fractions were identified, which were purified from 2-day-old soybeans and 4-day-old soybeans without or with flooding or drought for 2 days. Compared to 2-day-old soybeans, a number of 63, 74, and 40 ribosomal proteins were identified in control, flooding, and drought, respectively. Besides, protein abundance of most ribosomal proteins was decreased under stresses, indicating that protein synthesis might be suppressed under flooding and drought. Moreover, a number of 255, 368, and 103 proteins were predicated existing in endoplasmic reticulum in control, flooding, and drought, respectively. Based on functional analysis of endoplasmic reticulum proteins, protein-glycosylation and signaling were significantly changed under flooding and drought. Furthermore, proteins related to glycosylation were increased under flooding; however fewer proteins related to glycosylation were identified under drought. Additionally, proteins related to calcium signaling were significantly changed by flooding and drought. Taken together, these results suggest that protein synthesis were suppressed under both stresses; while protein glycosylation and calcium signaling might be different in soybean root tip under flooding and drought stresses.