The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, meaning that the human P2X7R (hP2X7R) is an attractive therapeutic target. The crystal structures of the zebrafish P2X4R in the closed and ATP-bound open states provide an unprecedented opportunity for structure-guided identification of new ligands. The present study performed virtual screening of ˜100,000 structurally diverse compounds against the ATP-binding pocket in the hP2X7R. This identified three compounds (C23, C40 and C60) out of 73 top-ranked compounds by testing against hP2X7R-mediated Ca2+ responses. These compounds were further characterised using Ca2+ imaging, patch-clamp current recording, YO-PRO-1 uptake and propidium iodide cell death assays. All three compounds inhibited BzATP-induced Ca2+ responses concentration-dependently with IC50s of 5.1 ± 0.3 μM, 4.8 ± 0.8 μM and 3.2 ± 0.2 μM, respectively. C23 and C40 inhibited BzATP-induced currents in a reversible and concentration-dependent manner, with IC50s of 0.35 ± 0.3 μM and 1.2 ± 0.1 μM, respectively, but surprisingly C60 did not affect BzATP-induced currents up to 100 μM. They suppressed BzATP-induced YO-PRO-1 uptake with IC50s of 1.8 ± 0.9 μM, 1.0 ± 0.1 μM and 0.8 ± 0.2 μM, respectively. Furthermore, these three compounds strongly protected against ATP-induced cell death. Among them, C40 and C60 exhibited strong specificity towards the hP2X7R over the hP2X4R and rP2X3R. In conclusion, our study reports the identification of three novel hP2X7R antagonists with micromolar potency for the first time using a structure-based approach, including the first P2X7R antagonist with preferential inhibition of large pore formation.