Osteosarcoma (OS) is the most frequently occurring pediatric bone malignancy in the world. Numerous miRNAs have previously been demonstrated to participate in the initiation and development of OS. The present study aimed to reveal the role of microRNA‑300 (miR‑300) in OS cells and elucidate the underlying mechanism involved. The expression of miR‑300 in the MG63 human OS cell line was monitored via quantitative polymerase chain reaction (qPCR). Following transfection with miR‑300 mimic, miR‑300 inhibitor or scramble control, MG63 cell viability, migration and apoptosis were respectively measured by 3‑(4,5‑dimethyl‑2‑thiazolyl)‑2,5‑diphenyltetrazolium bromide (MTT), modified two‑chamber migration assay and flow cytometry. Dual‑Luciferase reporter assays, qPCR and western blot analysis were subsequently performed to verify whether Twist1 was a direct target of miR‑300. Furthermore, the expression levels of nuclear factor (NF)‑κB pathway proteins were detected via western blot analysis. In MG63 cells, miR‑300 was effectively overexpressed or suppressed by transfection with miR‑300 mimic or inhibitor, respectively (P<0.001). Overexpression of miR‑300 significantly suppressed cell viability and migration, whereas it enhanced apoptotic rate (P<0.001). miR‑300 suppression exhibited contrary results (P<0.05, P<0.01 or P<0.001). Twist1 was demonstrated to act as a direct target of miR‑300, and was negatively regulated by miR‑300. In addition, miR‑300 overexpression downregulated the expression of the primary factors involved in the NF‑κB signaling pathway. These effects on OS cell proliferation and apoptosis may be due to the miR‑300 targeting of Twist1 and the suppressive effect on the NF‑κB signaling pathway.