•Nitrate bio-reduction was achieved in membrane biofilm reactors using propane or butane as electron donors.•Limited oxygen was an important triggering factor for propane/butane-driven nitrate reduction.•Propane/butane oxidizers (Mycobacterium/Rhodococcus/Thauera) and denitrifiers were enriched. Nitrate contamination has been commonly detected in water environments and poses serious hazards to human health. Previously methane was proposed as a promising electron donor to remove nitrate from contaminated water. Compared with pure methane, natural gas, which not only contains methane but also other short chain gaseous alkanes (SCGAs), is less expensive and more widely available, representing a more attractive electron source for removing oxidized contaminants. However, it remains unknown if these SCGAs can be utilized as electron donors for nitrate reduction. Here, two lab-scale membrane biofilm reactors (MBfRs) separately supplied with propane and butane were operated under oxygen-limiting conditions to test its feasibility of microbial nitrate reduction. Long-term performance suggested nitrate could be continuously removed at a rate of ∼40–50 mg N/L/d using propane/butane as electron donors. In the absence of propane/butane, nitrate removal rates significantly decreased both in the long-term operation (∼2–10 and ∼4–9 mg N/L/d for propane- and butane-based MBfRs, respectively) and batch tests, indicating nitrate bio-reduction was driven by propane/butane. The consumption rates of nitrate and propane/butane dramatically decreased under anaerobic conditions, but recovered after resupplying limited oxygen, suggesting oxygen was an essential triggering factor for propane/butane-based nitrate reduction. High-throughput sequencing targeting 16S rRNA, bmoX and narG genes indicated Mycobacterium/Rhodococcus/Thauera were the potential microorganisms oxidizing propane/butane, while various denitrifiers (e.g. Dechloromonas, Denitratisoma, Zoogloea, Acidovorax, Variovorax, Pseudogulbenkiania and Rhodanobacter) might perform nitrate reduction in the biofilms. Our findings provide evidence to link SCGA oxidation with nitrate reduction under oxygen-limiting conditions and may ultimately facilitate the design of cost-effective techniques for ex-situ groundwater remediation using natural gas. Display omitted