Abstract: The success of microbial enhanced oil recovery (MEOR) relies on complex microbial processes. Nevertheless, the contribution and mechanism of in-situ denitrification to microbial oil recovery remain unclear. In this study, eight denitrifying bacterial strains, designated T1, D1, D44, D46, D15, S1, S2 and S6, were isolated from the produced water of Xinjiang Oilfield, China, by a double layered plate method. The16S rDNA gene sequences of these denitrifying strains shared 100% similarity with Pseudomonas stutzeri (T1, D1, and D44), Pseudomonas putida (D46 and D15), and Pseudomonas aeruginosa (S1, S2, S6), respectively. The N₂O production effects of these strains on the physical properties of crude oil were evaluated with batch experiment. Results showed that the highest total gas yield was observed with sucrose as carbon source, and the maximal concentration of N₂O occurred with glycerol as carbon source. The denitrification process by these bacterial strains led to volume expansion and viscosity reduction of crude oil. Crude oil expansion rate was positively correlated with the concentration of N₂O, with a correlation coefficient of 0.983, but not correlated with the volume of total gas production. Strain S1, S2, and S6 produced 530-730 mg·L^-1 of surfactant using glycerol as ole carbon source, which could reduce surface tension and emulsify crude oil. However, these surfactant-producing strains produced less N₂O, exhibited weaker effects on oil swelling and viscosity reduction, compared to the none-surfactant-producing denitrifying strains. Our results suggested that more attention should be paid to the ability of N₂O production by denitrifying bacteria when exploiting microbial resources towards enhancing oil recovery.