Osteosarcoma (OS) is the most common primary bone malignancy in the adolescent population. Recent studies demonstrate that p38 gamma (p38γ) phosphorylates retinoblastoma (Rb) to promote cyclin expression, cell-cycle entry and tumorigenesis. Studying the potential function of p38γ in human OS, we show that p38γ mRNA and protein expression are significantly elevated in OS tissues and OS cells, whereas its expression is relatively low in normal bone tissue and in human osteoblasts/osteoblastic cells. Knockdown of p38γ in established (U2OS) and primary human OS cells potently inhibited cell growth, proliferation, migration and invasion, while promoting cell apoptosis. Furthermore, CRISPR/Cas9-induced p38γ knockout inhibited human OS cell progression in vitro. Conversely, ectopic overexpression of p38γ in primary human OS cells augmented cell growth, proliferation and migration. Signaling studies show that retinoblastoma (Rb) phosphorylation and cyclin E1/cyclin A expression were decreased following p38γ shRNA knockdown and knockout, but increased after ectopic p38γ overexpression. Collectively, these results show that p38γ overexpression promotes human OS cell progression.