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Response surface methodology was employed to enhance the anaerobic production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl. Glycerol is a promising carbon source used to anaerobically produce rhamnolipid. In a Plackett–Burman design, glycerol, KH2PO4 and yeast extract were significant factors. The proposed optimized medium contained the following: 46·55 g l−1 glycerol; 3 g l−1 NaNO3; 5·25 g l−1 K2HPO4·3H2O; 5·71 g l−1 KH2PO4; 0·40 g l−1 MgSO4·7H2O; 0·13 g l−1 CaCl2; 1·0 g l−1 KCl; 1·0 g l−1 NaCl; and 2·69 g l−1 yeast extract. Using this optimized medium, we obtained an anaerobic yield of rhamnolipid of 3·12 ± 0·11 g l−1 with a 0·85-fold increase. Core flooding test results also revealed that Ps. stutzeri Rhl grown in an optimized medium enhanced the oil recovery efficiency by 15·7%, which was 6·6% higher than in the initial medium. Results suggested that the optimized medium is a promising nutrient source that could effectively mobilize oil by enhancing the in situ production of rhamnolipid.Significance and Impact of the Study: The ex situ application of rhamnolipid for microbial enhanced oil recovery (MEOR) is costly and complex in terms of rhamnolipid production, purification and transportation. Compared with ex situ applications, the in situ production of rhamnolipid in anaerobic oil reservoir is more advantageous for MEOR. This study is the first to report the anaerobic production optimization of rhamnolipid. Results showed that the optimized medium enhanced not only the anaerobic production of rhamnolipid but also crude oil recovery.