Hypoxic-ischemic (HI) brain injury is associated with high mortality and severe long-term neurodevelopmental impairments in term newborns. Intranasal mesenchymal stem cell (MSC) therapy is a promising strategy to boost neurorepair after injury, and optimization strategies to further enhance its therapeutic potential are under development. In this study, we explored whether 24 h preconditioning of MSCs with 1000 ng/mL of osteopontin (OPN) could enhance MSC properties in vitro and in vivo. OPN-preconditioned MSCs (OPN-MSCs) showed increased activation of the ERK transcription pathway at 1 h during preconditioning and enhanced migration compared to naïve-MSCs. OPN preconditioning also altered gene expression of neurotrophic and immunomodulatory factors in MSCs. In vitro assessment of MSC potency showed that while OPN-MSCs were as effective as naïve-MSCs in reducing microglia activation, OPN preconditioning enhanced the potency of MSCs to boost neural stem cell differentiation into more complex neurons. However, in vivo, OPN-MSCs were not superior to naïve-MSCs in reducing lesion size in mice when applied at 3 days post-HI. Altogether, OPN preconditioning enhanced the migratory and neurotrophic properties of MSCs in vitro but not in vivo, highlighting its potential to optimize MSC function while underscoring the need for further research to refine in vivo translation and to evaluate functional outcomes for therapeutic efficacy.