T cell immunoglobulin domain and mucin domain‐containing molecule‐3 (Tim‐3), a member of the immunoglobulin superfamily, has been shown to play a crucial role in host adaptive immunity and tolerance. However, its role in kidney ischaemia–reperfusion injury (IRI) remains unknown. In this study, we investigated the role and mechanism of Tim‐3 signalling after kidney IRI. In an established murine model of kidney IRI, we found that Tim‐3 expression is enhanced on monocytes/macrophages. Anti‐Tim‐3 antibody RMT3‐23 ameliorates biochemical and histological kidney injury, reduces apoptosis and decreases macrophage infiltration and cytokine production in ischaemic kidneys. Cell culture experiments also demonstrated that the role of Tim‐3 in IRI‐induced macrophage activation leads to the secretion of proinflammatory cytokines and chemokines. In addition, Toll‐like receptor (TLR)‐4 and Nod‐like receptor (NLR) family CARD domain‐containing protein 4 (NLR‐C4) expression were enhanced after kidney IRI and decreased significantly by RMT3‐23. Tim‐3 not only promotes TLR‐mediated nuclear factor kappa B (NF‐κB) activation and cytokine and chemokine release, but also participates in NLR‐C4 inflammasome activation. Taken together, our data confirm that Tim‐3 signalling enhances injury after kidney IRI and demonstrated that Tim‐3 is involved in regulating TLR‐4/NF‐κB signalling and NLR‐C4 inflammasome activation, which provide evidence that Tim‐3 signalling is critical for kidney IRI and may provide a new means to ameliorate kidney tissue immune responses in the clinics.