High frequency and low intensity, diagnostic ultrasound methods are recognized to be safe in epidemiology and pathology but the bioeffects of these methods on molecular and proteomic levels are unknown. As a representative organism that can directly reflect the molecular response to stresses, Escherichia coli was selected for exposure to ultrasound probes C1-5, M5s and 9 L for 10 min and 20 min. ITRAQ was used to measure the expression of the cellular proteome. The results showed that both the frequency and time of exposure to ultrasound affected the proteome expression. Fifty biological processes were affected and nineteen metabolic processes, including carbohydrate metabolism, asparagine metabolism and phosphate import were differentially regulated. Lower frequency ultrasound caused copper export and iron‑sulfur cluster biosynthesis upregulation. Nine proteins (GlpD, AsnB, TdcB, CopA, IscR, IscU, IscS, IscA, RecA) were key for the adaption to ultrasound. Accordingly, the results of the potential risks based on the calculation of the orthologous genome clarified that relevant pathways and potentially sensitive individuals were worthy of further study. These findings offer insights into reveal the bioeffects of ultrasound at the metabolic network and proteomic levels. Copyright © 2020. Published by Elsevier B.V.