Objective To construct nucleotide-binding oligomerization domain-like receptors family member X1 (NLRX1) lentivirus plasmid and stable NLRX1-overexpressing A549 cell line. Methods Full-length cDNA encoding human NLRX1 was amplified from pCMV3-NLRX1 plasmid and then was inserted into pCDH-CMV-MCS-EF1-Puro vector to obtain NLRX1-overexpressing plasmid pCDH-NLRX1. pCDH-NLRX1 lentivirus particles were obtained by co-transfecting pCDH-NLRX1 with packaging plasmids (pMD2 and pAX2) into HEK293T cells. A549 cells were infected with concentrated pCDH-NLRX1 lentivirus particles and then were screened by puromycin to obtain stable NLRX1-overexpressed A549 cell line. The mRNA transcription and protein expression of NLRX1 were detected by real-time quantitative PCR, Western blot analysis and immunofluorescence. Results The lentivirus plasmid pCDH-NLRX1 was successfully constructed. After pCDH-NLRX1 and packaging plasmids were co-transfected into HEK293T cells, we obtained NLRX1 lentivirus particles with the titer of 1×105 TU/mL. After A549 cells were infected with lentivirus particles and screened by puromycin, a stable over-expression cell line of NLRX1 was obtained. NLRX1 was obviously expressed in the lentivirus-infected A549 cells and its mRNA and protein levels significantly increased. Conclusion NLRX1 lentivirus plasmid and stable NLRX1-overexpressing A549 cell line have been successfully constructed.