The present study determines the role of the Gab1 in hydrogen peroxide (H2O2)-induced death of human osteoblasts. We show that Gabl is required for H2O2-induced Akt activation to promote osteoblast survival. In OB-6 human osteoblasts, Gabl silencing (by targeted-shRNA) or complete knockout (by CRISPR-Cas9 KO plasmid) largely attenuated Akt activation by H2O2. Gab1-depleted OB-6 cells were more vulnerable to H2O2. Conversely, forced over-expression of Gabl by an adenovirus vector increased Akt activation to protect OB-6 cells from H2O2. Significantly, the anti-sense of microRNA-29a ("antagomiR29a") induced Gabl expression to facilitate H2O2-induced Akt activation, which protected OB-6 cells from apoptosis. AntagomiR-29a was however ineffective in Gab1-deficient and Akt-inhibited OB-6 cells. Forced over-expression of miR-29a induced Gab1 downregulation to inhibit H2O2-induced Akt activation, causing enhanced OB-6 cell death. miR-29a-induced actions were abolished by an adenovirus constitutively-active Aktl (Ad-caAkt1) in OB-6 cells. Together, microRNA-29a inhibition induces Gab1 upregulation and Akt activation to protect OB-6 osteoblasts from H2O2. (C) 2018 Elsevier Inc. All rights reserved.