Long non-coding RNA (lncRNA) NR2F1 antisense RNA 1 (NR2F1-AS1) has been reported to be an oncogene in several cancer types, including osteosarcoma (OS). However, the underlying fundamental molecular mechanism of NR2F1-AS1 in OS remains largely unknown, which the present study aimed to elucidate. The present study demonstrated that NR2F1-AS1 expression is markedly increased in OS, and NR2F1-AS1 was shown to exert oncogenic functions in OS. Further molecular mechanistic studies revealed that microRNA (miR)-485-5p and miR-218-5p were direct targets of NR2F1-AS1. More importantly, miR-485-5p and miR-218-5p exhibited low expression levels and were negatively correlated with NR2F1-AS1 expression in OS tissues. It was then identified that baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) was a direct target of miR-485-5p and miR-218-5p in OS cells. Furthermore, a series of experiments suggested that NR2F1-AS1 affects the proliferation, migration, invasion and apoptosis of OS cells by regulating BIRC5. Finally, it was revealed that silencing of NR2F1-AS1 repressed the OS cell malignant phenotype by binding with miR-485-5p and miR-218-5p, and then downregulating BIRC5 expression, which suggests that the NR2F1-AS1/miR-485-5p/miR-218-5p/BIRC5 axis could be a potential target for treating OS.