To explore the possibility of using lung perfusion single photon emission computed tomography (SPECT) scans to protect functional lung with optimizing intensity modulated radiotherapy (IMRT)plan in patients with stage III non-small cell lung cancer (NSCLC). 24 patients with stage III NSCLC who were candidates for radiotherapy were enrolled. All patients had PET-CT scans and SPECT scans. The two sets of images were accurately co-registered in the ADAC treatment planning system. SPECT images were used to define a volume of functioning lung (FL) and non-functioning lung (NFL). FL refers to the region of ≥30% maximum radioactive counts and the other region was non-functional lung (NFL). Then SPECT images were classified by comparing lung perfusion deficit with area of radiological abnormality. Grade 0: no lung perfusion deficit. Grade 1: the size of radiological abnormality was similar to the area of lung perfusion deficit. Grade 2: the area of lung perfusion was bigger than that of radiological abnormality, and extends to 1 pulmonary lobe. Grade 3: the area of lung perfusion deficit exceeds 1 pulmonary lobe. The optimized IMRT objective was to minimize the dose to FL. Statistical analysis was made for the difference between the two sets of IMRT plans. All patients had lung perfusion deficits, 8 patients with grade 1 damage, 6 patients with grade 2 damage, and 10 patient with grade 3 damage. After IMRT plan optimized, both the percentage of whole lung volume received dose ≥x Gy (WLVx)and the percentage of functional lung volume received dose ≥x Gy (FLVx) were decreased. WLV10, WLV15, WLV20, WLV25, WLV30 and WLV35 decreased 9.7%, 13.8%, 17.2%, 12.9%, 9.8% and 9.8%, while FLV10, FLV15, FLV20, FLV25, LV30 and FLV35 decreased 10.8%, 14.6%, 17.3%, 14.5%, 14.5% and 10.5%. Thus the FLVx was decreased more significantly. There are significant difference in WLV10, WLV15, WLV20, WLV25, WLV30 and FLV10, FLV15, FLV20, FLV25, FLV30 between the two sets of IMRT plans (p = 0.002, 0.000, 0.000, 0.005, 0.027 and 0.002, 0.000, 0.000, 0.006, 0.010). It was a good methodology for using SPECT-guidance to protect functional lung with optimizing intensity modulated radiotherapy in stage III non-small cell lung cancer patients, potentially reducing lung toxicity.