Tubulointerstitial inflammation plays a critical role in the progression of diabetic nephropathy (DN), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes contribute to renal interstitial inflammation in DN. Decreased expression of optineurin (OPTN) is also associated with the progression of DN. We investigated the role of OPTN in activation of the NLRP3 inflammasome in DN. We initially examined the renal biopsy tissues of 172 patients with type 2 DN and 32 nondiabetic patients with renal hamartoma. Expression of renal OPTN was significantly lower in patients with DN and negatively correlated with urinary levels of IL-1β and IL-18. Confocal microscopy analysis of the biopsies indicated no NLRP3 or IL-1β staining in OPTN-positive renal tubular epithelial cells (RTECs), indicating a negative correlation of OPTN expression with the activation of NLRP3 inflammasome. In vitro studies of murine RTECs indicated the levels of OPTN mRNA and protein decreased significantly after stimulation by a high glucose (HG) treatment. Relative to RTECs given HG, RTECs overexpressing OPTN showed significantly lower levels of NLRP3 expression, cleavage of caspase-1 and IL-1β, and release of IL-1 β and IL-18. Overexpression of OPTN in the presence of HG significantly increased the costaining of microtubule-associated protein 1A/1B-light chain 3-II and translocase of outer mitochondrial membrane 20 in RTECs, suggesting that OPTN enhances mitophagy. In addition, mitochondrial division inhibitor 1 blocked the inhibitory effect of OPTN overexpression on the activation of NLRP3 inflammasome in the presence of HG, indicating that OPTN overexpression inhibited NLRP3 inflammasome activation by enhancement of mitophagy. OPTN gene silencing significantly enhanced production of mitochondrial reactive oxygen species (mtROS) in the presence of HG. Compared with HG+OPTN small interfering RNA (siRNA)-treated RTECs, HG+OPTN siRNA+MitoTempo-treated RTECs attenuated NLRP3 inflammasome activation, suggesting that OPTN gene silencing activates the NLRP3 inflammasome by increasing mtROS in HG-treated RTECs. Taken together, our results demonstrate that OPTN inhibits the activation of NLRP3 inflammasome by enhancing mitophagy.-Chen, K., Feng, L., Hu, W., Chen, J., Wang, X., Wang, L., He, Y. Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy.