Transition-metal dichalcogenides (TMDs) have emerged in recent years as a special group of two-dimensional materials and have attracted tremendous attention. Among these TMD materials, molybdenum disulfide (MoS) has shown promising applications in electronics, photonics, energy, and electrochemistry. In particular, the defects in MoS play an essential role in altering the electronic, magnetic, optical, and catalytic properties of MoS, presenting a useful way to engineer the performance of MoS. The mechanisms by which lattice defects affect the MoS properties are unsettled. In this work, we reveal systematically how lattice defects and substrate interface affect MoS electronic structure. We fabricated single-layer MoS by chemical vapor deposition and then transferred onto Au, single-layer graphene, hexagonal boron nitride, and CeO as substrates and created defects in MoS by ion irradiation. We assessed how these defects and substrates affect the electronic structure of MoS by performing X-ray photoelectron spectroscopy, Raman and photoluminescence spectroscopies, and scanning tunneling microscopy/spectroscopy measurements. Molecular dynamics and first-principles based simulations allowed us to conclude the predominant lattice defects upon ion irradiation and associate those with the experimentally obtained electronic structure. We found that the substrates can tune the electronic energy levels in MoS due to charge transfer at the interface. Furthermore, the reduction state of CeO as an oxide substrate affects the interface charge transfer with MoS. The irradiated MoS had a faster hydrogen evolution kinetics compared to the as-prepared MoS, demonstrating the concept of defect controlled reactivity in this phase. Our findings provide effective probes for energy band and defects in MoS and show the importance of defect engineering in tuning the functionalities of MoS and other TMDs in electronics, optoelectronics, and electrochemistry.