Abstract Objective This study aimed to investigate the biological role of C5orf34 in Lung adenocarcinoma (LAD) and the mechanism of such role. Methods The mRNA expression of C5orf34 was analyzed using student's t -test based on the data obtained from TCGA database. Kaplan-Meier analysis was performed to evaluate the prognosis value of C5orf34. Chi-square (χ2) test was performed to analyze the correlation between C5orf34 expression and clinical features. The effect of C5orf34 siRNA on A549 and SPCA1 cells proliferation and migration were explored using CCK8 assay, wound healing assay and transwell assays, respectively. Western blot was performed to evaluate the expression of C5orf34, ERK, p-ERK, MEK and p-MEK. Results C5orf34 expression was enhanced in LAD and positively correlated with poor prognosis in patients with LAD. χ2 test revealed that C5orf34 expression was obviously associated with age (p = 0.016), Pathologic-Stage (p = 0.045) and Pathologic-N (p = 0.023). In addition, knockdown of C5orf34 significantly suppressed A549 and SPCA1 cells proliferation and motility (p < 0.01) in vitro. Moreover, by western blot analysis, we found that depleting C5orf34 remarkably reduced the ratio of p-MEK/MEK and p-ERK/ERK in A549 cells. Conclusion Our results firstly revealed that C5orf34 might play a facilitating role in LAD development and progression by regulating MAPK signaling pathway. Furthermore, our data implied that C5orf34 may be a potential predictor and treatment target for LAD. Highlights • C5orf34 expression was enhanced in LAD and predicted for poor prognosis. • Depleting C5orf34 expression significantly inhibited LAD cell proliferation. • Depleting C5orf34 expression significantly inhibited LAD cell migration and invasion. • Depleting C5orf34 suppressed MAPK signaling pathway activation. [ABSTRACT FROM AUTHOR]