Previous evidence has suggested that microRNAs (miRNAs or miRs), which belong to a class of non‑coding RNAs, shape cellular processes by regulating gene expression. Abnormal expression of miRNAs has been associated with tumorigenesis in multiple cancers. However, the function of miR‑336 in osteosarcoma (OS) remains unknown. The experimental procedures used in the present study included flow cytometry, reverse transcription‑quantitative polymerase chain reaction, luciferase reporter assay, invasion assay, western blot analysis and in vivo implantation. The results of the present study demonstrated that miR‑336 may serve as a tumor suppressor in OS. Downregulation of miR‑336 was observed in human OS specimens as well as OS cell lines. In addition, a significant negative correlation between sex determining region Y‑box 2 (Sox‑2) expression and miR‑336 was demonstrated. miR‑336 was confirmed to target the 3'‑untranslated region of Sox‑2 to inhibit proliferation, migration and invasion of OS cells. Consistently, restoration of Sox‑2 expression counteracted the effect of miR‑336, and recovered the tumorigenic potential of OS cells. The present study established a novel association between miR‑336 and Sox‑2 in OS. This relationship between miR‑336 and Sox‑2 may lead to improved knowledge concerning OS progression and sheds light on potential novel therapeutic interventions for OS treatment.