Engineered heme proteins posses excellent biocatalytic carbene N-H insertion results for sustainable synthesis and most of them have His as the Fe axial ligand. However, limited information of the basic reaction mechanisms is available and ground states of heme carbenes involved in the prior computational mechanistic studies are under debates. A comprehensive quantum chemical reaction pathway study was performed for the heme model with a His analogue as the axial ligand and carbene from the widely used precursor ethyl diazoacetate with the aniline substrate. The ground state of this heme carbene was calculated by the high level complete active space self-consistent field (CASSCF) approach, which shows a closed-shell singlet that is consistent with many experimental work. On this basis, the subsequent DFT calculations of ten main reaction pathways were compared. Results show that the most favorable pathway involves the initial formation of metal bound ylide, then exhibits a concerted rearrangement/dissociation transition state to form the free enol, which undergoes a water-assisted proton transfer process to yield the final N-H insertion product. This computational prediction was then validated by new experimental data using His-ligated myoglobin variants with different types of carbenes. Overall, this is the first comprehensive computational mechanistic study of heme carbene N-H insertions particularly for neutral His ligated heme proteins and the first high level CASSCF confirmation of the ground state of the used heme carbene. The experimental results are also the first in this field. Overall, these results build a solid basis for the proposed reaction mechanism to facilitate future biocatalytic carbene N-H insertion work.