The negative differential resistance (NDR) effect has been widely investigated for the development of various electronic devices. Apart from traditional semiconductor-based devices, two-dimensional (2D) transition metal dichalcogenide (TMD)-based field-effect transistors (FETs) have also recently exhibited NDR behavior in several of their heterostructures. However, to observe NDR in the form of monolayer MoS2, theoretical prediction has revealed that the material should be more profoundly affected by sulfur (S) vacancy defects. In this work, monolayer MoS2 FETs with a specific amount of S-vacancy defects are fabricated using three approaches, namely chemical treatment (KOH solution), physical treatment (electron beam bombardment), and as-grown MoS2. Based on systematic studies on the correlation of the S-vacancies with both the device's electron transport characteristics and spectroscopic analysis, the NDR has been clearly observed in the defect-engineered monolayer MoS2 FETs with an S-vacancy (VS) amount of ∼5 ± 0.5%. Consequently, stable NDR behavior can be observed at room temperature, and its peak-to-valley ratio can also be effectively modulated via the gate electric field and light intensity. Through these results, it is envisioned that more electronic applications based on defect-engineered layered TMDs will emerge in the near future.