We investigated the mechanism of action of photobiomodulation (PBM) with light‐emitting diode (led) 640 nm of glutamine‐dependent CT26 cells. Cells were exposed to 0.147–10.979 mW/cm2 of 640 ± 15 nm laser light for 15 min/day for 10 days. Cell proliferation and apoptosis were detected by MTT (3‐(4,5)‐dimethylthiahiazo (‐z‐y1)‐3,5‐diphenytetrazoliumromide) and annexin V‐FITC assays. mRNA and protein levels of cell proliferation‐related genes were measured by RT‐PCR and western blotting, respectively. With Gln 7.94 mM, on Day 8 and 10, genes GLUT1, MEK1, ERK2, BCL2, E2F1, HO‐1, Ctnnb1, and Per2 was significantly upregulated (p < 0.01) of glutamine addiction. In PBM therapy, compared with the non‐illuminated group, 2.17 mW/cm2 can significantly reduce cell apoptosis, the mRNA level of gene mTOR1 was significantly upregulated, and the protein level of raptor of GLUT1 and mTOR1, MEK1/2, and ERK1/2 were upregulated. LED 640 nm inhibits cell apoptosis without increasing cell proliferation by regulating GLUT1, MEK/ERK, and PI3K/AKT/mTOR signals.