Blakeslea trispora is commercially used as natural source of beta-carotene in industry. We found that ethylene treatment (ET) can promote the production of beta-carotene in B. trispora by 52 +/- 0.20%, reaching 1.87 +/- 0.11 g/L. However, the metabolic responses to ethylene in B. trispora are not fully understood. In this work, gas chromatography/mass spectrometry-based metabolomics approach and two-dimensional electrophoresis-based proteomics approach by MALDI-TOF/TOF were employed to gain a better understanding of its metabolisms. We found that 31 metabolites and 9 proteins were directly related to the enhancement of beta-carotene production in combination with the partial least-squares-discriminant analysis and two-dimensional electrophoresis analysis. Those metabolites and proteins were involved in tricarboxylic acid (TCA) cycle, carbohydrate metabolism, amino acid metabolism, fatty acid metabolism and mevalonate pathway. Our results show that ethylene causes the decrease of TCA flux and the accumulation of acetyl-CoA, which contributes to the enhancement of beta-carotene production. As responses to ethylene, transcription of key genes involved in mevalonate pathway (including hmgR, ipi, carG, carRA and carB) was induced by ethylene, resulting in the enhancement of beta-carotene production. (C) 2017 Elsevier Ltd. All rights reserved.