A series of new [1,2,3]triazolo[4,5-d]pyrimidine/thiourea hybrids were designed and synthesized through the scaffold replacement/ring cleavage strategy. SARs studies revealed that the N-heteroarene moiety attached to the thiourea is preferred over the phenyl ring for the R2 substituents, while the hydrophobic aromatic group is beneficial for improving the activity. Among these compounds, compound 5r significantly inhibited cell growth of lung cancer cell lines H1650 and A549 (IC50 = 1.91, 3.28 μM, respectively), but was less toxic against the normal cell line GES-1 (IC50 = 27.43 μM). Mechanistic studies showed that compound 5r could remarkably inhibit the colony formation of H1650 cells, induced apoptosis possibly through the intrinsic apoptotic pathways, and arrested the cell cycle at G2/M phase. Our studies suggest that the [1,2,3]triazolo[4,5-d]pyrimidine/thiourea hybrids are a new class of chemotypes possessing interesting antiproliferative activity against lung cancer cells and could be potentially utilized for designing new antitumor agents.