Background: Campylobacter jejuni thrives in the stomach's acidic environment. Two proteins, Cj0414 and Cj0415, orthologs of gluconate dehydrogenase (GADH), were previously identifed in C. jejuni. This study aimed to determine the role of Cj0414 in Campylobacter acid resistance and virulence. Methods: C. jejuniΔcj0414 deletion mutant was constructed and tested alongside the wild type (wt) and com- plemented strains for stress survival and host pathogenesis. The role of D-gluconate in Campylobacter growth was characterized spectrophotometrically. The contribution of Cj0414 to Campylobacter acid survival was assessed in Mueller Hinton (MH) medium acidifed by HCl. The importance of Cj0414 for Campylobacter survival to oxidative stress was evaluated by the disc inhibition assay. Assessment of bacterial adherence, invasion and intracellular survival were conducted using HCT116 cells. Finally, the virulence of C. jejuni strains (wt, Δcj0414 mutant and C. jejuni Δcj0414+cj0414) was characterized in vivo using G. mellonella. Results: There was no difference (P > 0.05) in bacterial growth for C. jejuni wt, Δcj0414 mutant and Δcj0414+cj0414 complemented strain either in absence or presence of gluconate. The capacity of C. jejuni Δcj0414 mutant to survive acid was impaired as compared to C. jejuni wt. The acid survival of C. jejuni Δcj0414 mutant signifcantly reduced at 5, 10 and 15 min following acid exposure (8.51 % ± 3.7, 0.4 % ± 0.05 and 0.2 % ± 0.005, respectively) relative to C. jejuni wt (48.9 % ± 0.6, 25.8 % ± 1.3 and 1.9 % ± 0.14, respectively). Furthermore, C. jejuni Δcj0414 mutant showed impaired adherence, invasion, and replication within eukaryotic cells. Regarding in vivo G. mellonella infection, there was a signifcant difference between C. jejuni Δcj0414 that killed only 50% of larvae relative to the wild type that killed all larvae. Importantly, cj0414 complementation markedly restored Δcj0414 mutant capacity to kill G. mellonella larvae. Conclusions: Cj0414 is essential for C. jejuni's acid survival and virulence and consequently could be a potential therapeutic target for combating this enteric pathogen. This study advances insights into C. jejuni pathogenesis, informing strategies to control C. jejuni infections.