Nanoscale devices made using two-dimensional (2D) materials, including transition-metal dichalcogenides (TMDs) such as molybdenum disulfide (MoS2), are currently being explored for various applications, e.g., optoelectronic devices, water desalination membranes, and DNA sequencing. In such applications, defects and nanopores play a key role in modulating the 2D material’s physicochemical properties, e.g., the band gap, chemical reactivity, catalytic activity, and molecular/ionic permeation rate. However, there exists a lack of a fundamental understanding of nanopore formation in TMDs, especially MoS2. In this work, we elucidate the mechanism of nanopore formation in 2D MoS2 using an extensive set of first-principles density functional theory (DFT) calculations. We calculate the energies of etching of atoms using DFT and use Marcus theory for atom-transfer reactions to convert the energies to activation barriers. We postulate the role of silicon as an etchant and show that the MoS6 vacancy can be a potential nucleation site for the growth of nanopores in MoS2. By fitting our kinetic predictions to experimental microscopy data on the formation time of a MoS6 vacancy, we show that the intrinsic barrier for the etching of atoms in MoS2, as described by Marcus theory, is 0.228 eV in the presence of silicon atoms. We find that the slowest step in the formation of the MoS6 vacancy is the step leading from a S5 vacancy to a S6 vacancy, with an activation barrier of ∼0.85 eV. This allows atoms to be etched at close to room-temperature conditions (27 °C) under the action of an electron beam that can move around the silicon etchant atoms. Using this new understanding of the nanopore formation process, researchers will be able to accurately model the controlled fabrication of nanopores in 2D MoS2.