Activation of the nod-like receptor protein 3 (NLRP3)-inflammasome is required for IL-1β release and is a key component of obesity-induced inflammation and insulin resistance (IR). This study hypothesized that supplementation with a casein hydrolysate (CH) would attenuate NLRP3-inflammasome mediated IL-1β secretion in adipose tissue (AT) and improve obesity-induced IR. J774.2 macrophages were LPS primed (10ng/ml) and stimulated with adenosine triphosphate (ATP) (5mM) to assess NLRP3 inflammasome activity. Pre-treatment with CH (1mg/mL; 48h) reduced caspase-1 activity and decreased IL-1β secretion from J774.2 macrophages in vitro. 3T3-L1 adipocytes cultured with conditioned media from CH pre-treated J774.2 macrophages demonstrated increased phosphorylated (p)AKT expression and improved insulin sensitivity. C57BL/6JOLaHsd mice were fed chow or high fat diet (HFD) for 12 weeks ± CH resuspended in water (0.5% w/v). CH supplementation improved glucose tolerance in HFD-fed mice as determined by glucose tolerance test. CH supplementation increased insulin-stimulated pAKT protein levels in AT, liver and muscle after HFD. Cytokine secretion was measured from AT and isolated bone marrow macrophages (BMM) cultured ex vivo. CH supplementation attenuated IL-1β, tumor necrosis factor (TNF) α and IL-6 secretion from AT and IL-1β, IL-18 and TNFα from BMM following ATP stimulation ex vivo. This novel CH partially protects mice against obesity-induced hyperglycemia coincident with attenuated IL-1β secretion and improved insulin signaling. This article is protected by copyright. All rights reserved.