A pro-inflammatory environment is characteristic of obesity and polycystic ovary syndrome (PCOS). This environment through cytokines secretion negatively affects insulin action. Endometria from women with both conditions (obesity and PCOS) present high TNF-α level and altered insulin signaling. In addition, these patients present reproductive failures that could be associated to an abnormal endometrial function. Here, TNF-α and IL-6 effects on insulin signaling pathway were evaluated. Serum and endometrial IL-6, phospho-IRS1-S270 (inactive form) and phospho-IRS1-Y612 (active form) levels were evaluated in women with: Normal-Weight, Obesity and Obesity-PCOS. In endometrial cells under hyperandrogenic/hyperinsulinic conditions resembling PCOS, it was evaluated IL-6/TNF-α effects on phospho-IRS1-S270, phospho-IRS1-Y612, phospho-AKT-S473 levels, and S6K and JNK activation (IRS1-inactivating molecules). In obesity groups, diminution of IRS1-active form was observed, being more significantly in Obesity-PCOS; whereas, IRS1-inactive form increased in Obesity-PCOS. Serum and endometrial IL-6 were higher in Obesity-groups compared to Normal-Weight. In endometrial cells, TNF-α increases phospho-IRS1-S270, while IL-6 decreases phospho-IRS1-Y612. Importantly, TNF-α and IL-6 promote S6K and JNK activation; TNF-α increases and IL-6 decreases phospho-AKT-S473 levels. Thus, pro-inflammatory cytokines in endometrium could negatively influence insulin signaling by different mechanisms: TNF-α promotes activation of IRS1-inactivating kinases, whereas, IL-6 decreases IRS1 and AKT activation. Moreover, when obesity and PCOS are present the disruption of insulin signaling is aggravated. These effects could explain endometrial abnormal function and reproductive failures observed in women with obesity and PCOS.