Aims Rahnella aquatilis is a Gram-negative bacterial species with potential for agricultural and industrial applications, as well as a human pathogen. This study aims to identify an optimal reference gene to explore antibiotic resistance and stress tolerance in R. aquatilis using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Methods and Results Expression levels of 14 housekeeping genes in R. aquatilis were estimated by RT-qPCR under six different conditions: exponential phase, stationary phase, acid, salinity, antibiotic and oxidative stresses. BestKeeper and the Delta Ct method were used to evaluate the stability of each gene. The atpD gene was stably expressed in all conditions, thus was selected and validated as an optimal reference gene. Transcript levels of 17 putative ampicillin-resistance genes in R. aquatilis strain HX2 were evaluated using the proposed RT-qPCR. Six genes encoding efflux transporters and beta-lactamase were overexpressed after ampicillin treatment. Additionally, the expression of seven putative stress response genes in strain HX2 was assessed, and five genes were up-regulated by respective stress treatments. Conclusions The atpD gene has been identified as an optimal reference gene for expression analysis of R. aquatilis responses to abiotic stresses by RT-qPCR. Significance and Impact of the Study The proposed RT-qPCR is suitable for gene expression analysis in R. aquatilis, thus useful for studying antimicrobial resistance and stress tolerance in this bacterium and others closely related.