n-Caproic acid (CA) is gaining increased attention due to its high value as a chemical feedstock. Ruminococcaceae bacterium strain CPB6 is an anaerobic mesophilic bacterium that is highly prolific in its ability to perform chain elongation of lactate to CA. However, little is known about the genome-wide transcriptional analysis of strain CPB6 for CA production triggered by the supplementation of exogenous lactate. In this study, cultivation of strain CPB6 was carried out in the absence and presence of lactate. Transcriptional profiles were analyzed using RNA-seq, and differentially expressed genes (DEGs) between the lactate-supplemented cells and control cells without lactate were analyzed. The results showed that lactate supplementation led to earlier CA p,roduction, and higher final CA titer and productivity. 295 genes were substrate and/or growth dependent, and these genes cover crucial functional categories. Specifically, 5 genes responsible for the reverse β-oxidation pathway, 11 genes encoding ATP-binding cassette (ABC) transporters, 6 genes encoding substrate-binding protein (SBP), and 4 genes encoding phosphotransferase system (PTS) transporters were strikingly upregulated in response to the addition of lactate. These genes would be candidates for future studies aiming at understanding the regulatory mechanism of lactate conversion into CA, as well as for the improvement of CA production in strain CPB6. The findings presented herein reveal unique insights into the biomolecular effect of lactate on CA production at the transcriptional level.