Background MicroRNAs are known to regulate carcinogenesis of osteosarcoma. Although, miR-16-5p is known to exert inhibitory effects on several forms of cancers, its effects on the growth and invasion of osteosarcoma have not been studied. Methods We collected human osteosarcoma specimens and adjacent tissues to detect the expression of miR-16-5p by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. The proliferation, migration, and invasion of MG63 and HOS cells following miR-16-5p overexpression and inhibition were detected with cell counting kit-8, wound healing assay, and Transwell assay, respectively. An expression vector carrying a mutated 3 '-untranslated region of mothers against decapentaplegic homolog 3 (Smad3) was constructed. Results The results showed that miR-16-5p expression was downregulated in osteosarcoma tissues and cells as compared with adjacent counterparts, while Smad3 was overexpressed in osteosarcoma cells. The overexpression of miR-16-5p resulted in the inhibition of the proliferation, migration, and invasion of osteosarcoma cells and enhanced the therapeutic effect of cisplatin. These effects were attenuated with miR-16-5p expression inhibition. In cells transfected with miR-16-5p mimic, Smad3 expression decreased, while this effect was absent in the cells carrying mutated Smad3. Conclusions Therefore, miR-16-5p inhibits the growth and invasion of osteosarcoma by targeting Smad3.