Long non-coding RNAs (lncRNAs) have a role in the occurrence and development of lung squamous cell carcinoma (LUSC). lncRNA gamma-butyrobetaine hydroxylase 1 (BBOX1)-antisense 1 (AS1) may contribute to disease development. However, there are no studies on the role of BBOX1-AS1 in LUSC to date. In the present study, an in-house gene microarray analysis was performed to detect the differentially expressed lncRNAs and mRNAs between three pairs of LUSC and normal lung tissues. Only one lncRNA, BBOX1-AS1, was differentially expressed in the in-house microarray and The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and ArrayExpress databases. Reverse transcription-quantitative PCR (RT-qPCR) was then performed and the original RNA-sequencing data from the TCGA, GEO and ArrayExpress datasets were used to determine the expression and clinical value of BBOX1-AS1 in LUSC. In addition, a Cell Counting Kit-8 assay, cell cycle analysis and scratch assay were performed to explore whether BBOX1-AS1 expression affected the proliferation and migration of LUSC cells in vitro. The results of the RT-qPCR analysis and data obtained from the TCGA database, GEO datasets, in-house gene microarray and standard mean deviation analysis all supported the upregulated expression level of BBOX1-AS1 in LUSC. Furthermore, silencing of BBOX1-AS1 inhibited the proliferation and migration of LUSC cells according to in vitro assays. In addition, the cells were arrested in S-phase after knockdown of BBOX1-AS1. In conclusion, the expression level of BBOX1-AS1 was upregulated in LUSC tissues. BBOX1-AS1 may exert an oncogenic effect on LUSC by regulating various biological functions. However, additional functional experiments should be performed to verify the exact mechanism.