Colorectal cancer is a common cancer with metachronous distant metastases still threatening overall survival. Tumor oxygen level influences tumor radiosensitivity in relation to autophagy and apoptosis. The objective of this study is to evaluate the expression and interaction between multiple key regulators in different oxygen levels. Human colorectal adenocarcinoma HT-29 cells were cultured in 1% or 10% oxygen level and irradiated by 2 Gy with different incubation time. Autophagy key regulators, AMPK, HIFs and JNK were evaluated by Western blot. Sequential autophagy key regulator activation was observed in the order of AMPK, HIF-1 alpha, HIF-2 alpha and JNK. 10% oxygen level could promote autophagy with similar degree of autophagy activation as 1% oxygen level in 48-h while irradiation could slightly inhibit autophagy. The results of this study supported prior evaluation of oxygen level and autophagy regulators for improving treatment efficacy and indicated the possible directions in developing individualized radiotherapy by selective targeting of hypoxic regions. (C) 2019 The Authors. Published by Elsevier Inc.