Evidence suggests that both 14-3-3σ and long non-coding RNA HOX transcript antisense RNA (HOTAIR) are involved in the tumorigenesis and progression of lung cancer. In the present study, the potential association between 14-3-3σ and HOTAIR in non-small cell lung cancer (NSCLC) was investigated. In tissue samples collected from 54 patients with NSCLC, expression of HOTAIR and 14-3-3σ was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). After stable ectopic expression of HOTAIR and stable HOTAIR knockdown in PC9 cancer cells, the effect of HOTAIR on levels of mRNA and protein 14-3-3σ expression levels were detected using RT-qPCR and western blotting, respectively. Expression of HOTAIR and 14-3-3σ in NSCLC tissues was significantly higher than in adjacent non-cancerous lung tissue (P<0.05). Correlation analysis also identified a correlation between levels of HOTAIR and 14-3-3σ expression in NSCLC tissues (r=0.725, P=0.0005). In addition, overexpression and knockdown of HOTAIR in the human NSCLC cell line PC9 led to the upregulation and downregulation of 14-3-3σ, respectively, at both the mRNA and protein levels (all P<0.05). To the best of our knowledge, the present study provides the first in vivo and in vitro evidence to suggest that HOTAIR promotes the expression of 14-3-3σ in NSCLC. The potential association between HOTAIR and 14-3-3σ indicates that both biomolecules may be viable targets in anticancer therapy.