BACKGROUND The aim of this study was to investigate the expression of a novel long noncoding RNA (lncRNA), LL22NC03-N64E9.1, and its effect on the phenotype of lung cancer cells and tissues using The Cancer Genome Atlas (TCGA) RNA sequencing data and other publicly available profiling data. MATERIAL AND METHODS The lung cancer dataset GSE30219 was downloaded from the Gene Expression Omnibus (GEO) repository. Differentially expressed lncRNA, LL22NC03-N64E9.1, in 48 lung cancer tissue samples and adjacent normal lung tissues, normal lung cell lines BEAS-2B and A549, and lung cancer cell lines, H1703, and H292, were detected by quantitative reverse transcription polymerase chain reaction (PCR) (qRT-PCR). Interference efficiency was performed using small interfering RNA (siRNA). Tumor levels of lncRNA, LL22NC03-N64E9.1, and clinicopathological parameters were statistically analyzed. RESULTS Analysis of the GSE30219 test cohort showed that lncRNA, LL22NC03-N64E9.1 expression was significantly increased in lung cancer. In clinical tissue samples, the level of LL22NC03-N64E9.1 in patients with lung cancer was significantly increased compared with adjacent normal lung tissues (P<0.001). The level of LL22NC03-N64E9.1 in patients with lung cancer was significantly correlated with tumor size and TNM stage (P<0.05), but not with age, sex and the presence of lymph node metastasis (P>0.05). In the H292 cells, following knockdown of LL22NC03-N64E9.1, cell proliferation and cloning were reduced. CONCLUSIONS Expression of lncRNA, LL22NC03-N64E9.1, promoted proliferation of lung cancer cells in vitro, was highly expressed in lung cancer tissues and was associated with increased overall survival (OS), tumor size, and tumor stage in patients with lung cancer.