Sterile inflammation (SI) is a common pathology in numerous diseases, as a cause or consequence. Hypothyroidism (HT) is an inflammatory condition with complications stemming from excess cytokine release. A thyroidectomy-induced HT rat model was used to evaluate the impact of human umbilical cord blood–derived mesenchymal stem cells (hUCB-MSCs) on splenic inflammation and histoarchitecture. Thirty male Wistar rats were allocated equally as Sham, HT and HT+hUCB-MSCs groups. HT was confirmed by serum-free thyroxin 3, thyroxin 4 and thyroid stimulating hormone levels. Splenic homogenates were analyzed for oxidative stress and inflammation markers. Histopathological and immunohistochemical analyses were conducted to assess splenic stromal and parenchymal elements, high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), pro-inflammatory cytokine immune expression, in addition to the innate and adaptive immune cell populations and their proliferative and apoptotic activities. Correlations between HMGB1 level and various parameters were evaluated. hUCB-MSCs significantly downregulated HMGB1, TLR4 and pro-inflammatory cytokine immune expression and attenuated immune cell activation through increasing antioxidant status, attenuating lipid peroxidation, and modulating cellular proliferation and apoptosis, thus protecting the spleen against HT-induced structural damage and immune cell overactivation. Collectively, our results demonstrate the therapeutic efficacy of hUCB-MSCs in mitigating HT-induced SI.