Alzheimer's disease (AD) is considered a prevalent neurological disorder with a neurodegenerative nature in elderly people. Oxidative stress and neuroinflammation due to amyloid β (Aβ) peptides are strongly involved in AD pathogenesis. Klotho is an anti-aging protein with multiple protective effects that its deficiency is involved in development of age-related disorders. In this study, we investigated the beneficial effect of Klotho pretreatment at different concentrations of 0.5, 1, and 2 nM against Aβ1-42 toxicity at a concentration of 20 μM in human SH-SY5Y neuroblastoma cells. Our findings showed that Klotho could significantly and partially restore cell viability and decrease reactive oxygen species (known as ROS) and improve superoxide dismutase activity (SOD) in addition to reduction of caspase 3 activity and DNA fragmentation following Aβ1-42 challenge. In addition, exogenous Klotho also reduced inflammatory biomarkers consisting of nuclear factor-kB (NF-kB), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in Aβ-exposed cells. Besides, Klotho caused downregulation of Wnt1 level, upregulation of phosphorylated cyclic AMP response element binding (pCREB), and mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) with no significant alteration of epsilon isoform of protein kinase C (PKCε) after Aβ toxicity. In summary, Klotho could alleviate apoptosis, oxidative stress, and inflammation in human neuroblastoma cells after Aβ challenge and its beneficial effect is partially exerted through appropriate modulation of Wnt1/pCREB/Nrf2/HO-1 signaling.