Although conventional clinical treatment with low LET (linear energy transfer) including gamma-ray and X-ray has been widely used for radiotherapy in various cancers, however, ineffective outcomes occur due to radioresistance caused by p53 mutation. High LET has become alternative since it is able to induce apoptosis regardless of p53 status. Indeed, the molecular mechanisms toward high LET have been suggested. Nevertheless, most studies have been done in monolayer culture system which cannot promptly represent solid tumor microenvironment. Here we applied in vivo mimic 3D spheroid to conduct microarray-based genomic expression and molecular signaling pathway analyses under neutron irradiation. As a result, 3D spheroid system was achieved using thermorevesible gel system. An effective apoptosis-inducible dose of neutron was determined by Acridine Orange (AO) staining in 3D spheroid. Differentially expressed genes in both unique and common responses to neutron were identified in the 3D spheroid compared to the monolayer cells. Total 95 and 169 genes were notably altered at transcription level toward neutron in monolayer and 3D spheroid system, respectively. Based on microarray data, putative apoptosis signaling was depicted using Pathway Studio software. In 3D-in vivo mimic model, the molecular networks interacted with ITGB1, MAP4K4, PAPPA, and SGK1 might be suggested as plausible molecular pathways. In conclusion, we demonstrate novel molecular signaling and corresponding targets of in vitro solid tumor following high LET exposure. This result might provide critical clues for clarification of neutron-induced apoptosis mechanism.