Abstract A lithium substituted 58S bioactive glass (BG) containing from 0 to 10 mol% Li 2 O was synthesized through the sol-gel technique and the effect of Lithium substitutions on in vitro hydroxycarbonate apatite formation, osteoblast-like cell responses and antibacterial efficiency was studied. Structural and morphological evaluation using X-ray diffraction analysis, Fourier transform infrared spectroscopy, scanning electron microscopy revealed that 58S BG with 5 mol% Li 2 O (BG-5) had no significant effect on the formation rate of crystalline hydroxycarbonate apatite however 58S BG with 10 mol% Li 2 O lowered the bioactivity of the BG. The lower bioactivity of BG-10 was associated with a lower rate of Si and Ca ion release into the simulated body fluid. The 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and alkaline phosphatase (ALP) assessment methods revealed that the presence of Li had a stimulating influence on increasing of both proliferation and differentiation of MC3T3-E1 cells. A qualitative assessment of cell viability with Live/Dead technique and visualization of the morphology of actin fibers and nuclei of MC3T3 cells treated with Li-BGs with DAPI/Actin staining techniques showed that addition of Li resulted in an increase in the mean number of DAPI-labeled nuclei which was in reasonable agreement with MTT test. The Li substituted BGs exhibited a potential antibacterial effect against methicillin-resistant Staphylococcus aureus bacteria (MRSA). Results suggest that sample BG-5 is a good candidate for bone tissue engineering with optimal cell proliferation and ALP activity, good bioactivity and significant high antibacterial potential against MRSA.