In 2D semiconductors, doping offers an effective approach to modulate their optical and electronic properties. Here, an in situ doping of oxygen atoms in monolayer molybdenum disulfide (MoS2) is reported during the chemical vapor deposition process. Oxygen concentrations up to 20–25% can be reliable achieved in these doped monolayers, MoS2‐xOx. These oxygen dopants are in a form of substitution of sulfur atoms in the MoS2 lattice and can reduce the bandgap of intrinsic MoS2 without introducing in‐gap states as confirmed by photoluminescence spectroscopy and scanning tunneling spectroscopy. Field effect transistors made of monolayer MoS2‐xOx show enhanced electrical performances, such as high field‐effect mobility (≈100 cm2 V−1 s−1) and inverter gain, ultrahigh devices’ on/off ratio (>109) and small subthreshold swing value (≈80 mV dec−1). This in situ oxygen doping technique holds great promise on developing advanced electronics based on 2D semiconductors. In situ oxygen substitutional doping in MoS2 monolayers is achieved by a one‐step chemical vapor deposition process and lateral heterostructures with controlled doping concentrations are reliably fabricated. The bandgap tuniability (from 2.25 to 1.75 eV) and n‐doping electronic properties are relized. This nondestructive oxygen doping technique holds great promise on future electronics based on 2D semiconductors.