To find out an efficient and no second pollution method of degradation oxytetracycline (OTC), the effective reasons of γ-ray absorbed dose, initial concentration of OTC, and pH value were studied. The results showed that γ-ray irradiation can more effectively degrade OTC in aqueous solution with higher absorbed dose, lower OTC aqueous solution concentration, and lower pH value. OTC and five main degraded products (m/z of 447, 443, 433, 279, and 218) were identified by liquid chromatography-mass spectrometry (LC-ESI-MS). Gaussian 03 program calculated molecule energy, Natural Population Analysis and bond rate of OTC and its degradation products. Result shows that there are two main pathway of OTC degradation. The first pathway: three methyl groups on N(27) and C(10) degraded firstly, product m/z 279 break from C(12)−C(15) and C(13)−C(18) secondly, and molecule m/z 279 departed at C(10) and a dehydrated molecule m/z 261 is found finally. The second pathway: dehydration and rearrangement are happened; then break at C(13)−C(14) and a thickening rings m/z 230 is formed; at last, carbon ring embed with oxygen degraded CH2 and COOH. LC-ESI-MS detection combining with Gaussian 03 calculation is an efficient and accurate method to find out the degradation pathway of OTC.Cited