Abstract: Objective To clarify the specific mechanism of oncogenic B-Raf^V600E presenting resistance to the auto-regulatory negative feedback loop of Mps1 and B-Raf^WT/MEK/ERK signaling pathway. Methods (1) Sbcl2 cells were transfected with B-Raf^WT or Mps1-KD and the activity of p-ERK was analyzed by Western blot. (2)The p-ERK levels were detected by Western blot in SK-MEL31, Sbcl2 and WM35 cells which harbor B-Raf^WT, meanwhile the expression of p-ERK was also detected in SK-MEL28 and A375 cells which harbor B-Raf^V600E. (3) AKT was knocked down in SK-MEL31, Sbcl2 and WM35 cells with B-Raf wild-type genetic background, then transfected with Mps1, and the activity of p-ERKwas analyzed by Western blot. (4) The endogenous wild-type B-Raf in Sbcl2, WM35 and SK-MEL31 cells were knocked down, then exogenous B-Raf^V600E was transfected into the cells. The expression of p-AKT was detected by Western blot. Meanwhile we knocked down B-Raf^V600E in A375 and SK-MEL-28 melanoma cells for assessing the expression of p-AKT. Results (1) The auto-regulatory negative feedback loop of Mps1 on the B-Raf^WT/MEK/ERK signaling pathway was independent of kinase activity of Mps1. (2) The expression of exogenous Mps1 could enhance AKT phosphorylation and suppress ERK activity in melanoma cells harboring B-RafWT but not B-Raf^V600E. (3) Knockdown of AKT in different melanoma cell lines with B-Raf wild-type genetic background leaded to Mps1 losing the ability of regulating the feedback loop. (4) AKT phosphorylation was suppressed by B-Raf^V600E, which contributed to the abrogation of the regulatory negative feedback loop of Mps1 on the MAPK signaling pathway. Conclusion The auto-regulatory negative feedback loop of Mps1 on the B-RafWT/ ERK signaling pathway is independent of kinase activity of Mps1. Oncogenic B-Raf^V600E could surppress AKT phosphorylation to abrogate the regulatory negative feedback loop of B-Raf/MEK/ERK/Mps1.