Cancer recurrence is often associated with the acquisition of radioresistance by cancer tissues due to failure in radiotherapy. The underlying mechanism leading to the development of acquired radioresistance in the EMT6 mouse mammary carcinoma cell line and the potential pathway involved was investigated by comparing differential gene expressions between parental and acquired radioresistance cells. EMT6 cell line was exposed to 2 Gy/per cycle of gamma-ray and the survival fraction between EMT6-treated and parental cells was compared. EMT6 (acquired radioresistance) cells was developed after 8 cycles of fractionated irradiation. The development of EMT6 cells was confirmed with further irradiation at different doses of gamma-ray, and both the survival fraction and migration rates were measured. Higher survival fraction and migration rates were obtained in EMT6 cells after exposure to 4 Gy and 8 Gy gamma-ray irradiations compared to their parental cells. Gene expression between EMT6 and parental cells was compared, and 16 genes identified to possess more than tenfold changes were selected and validated using RT-PCR. Out of these genes, 5 were significantly up-regulated i.e., IL-6, PDL-1, AXL, GAS6 and APCDD1. Based on pathway analysis software, the development of acquired radioresistance in EMT6 was hypothesized through JAK/STAT/PI3K pathway. Presently, CTLA-4 and PD-1 were determined to be associated with JAK/STAT/PI3K pathway, where both their expressions were significantly increased in EMT6 compared to parental cells in the 1st, 4th and 8th cycle of radiation. As a conclusion, the current findings provided a mechanistic platform for the development of acquired radioresistance in EMT6 through overexpression of CTLA-4 and PD-1, and novel knowledge on therapeutic targets for recurrent radioresistant cancers.