Structural defects play an important role in the optimization of material structures and properties, especially in low-dimensional systems such as two-dimensional (2D) materials. In this work, we investigated the formation, aggregation, and diffusion of vacancy defects in atomically thin black phosphorus (BP) via in situ high-resolution transmission electron microscopy. Vacancy defects including di-vacancies (DVs), vacancy clusters (e.g. tetra-vacancy and TV), and vacancy lines were confirmed as the primary forms of structural defects in BP. DV and TV defects were found to be highly mobile. The defects preferentially diffused and migrated along the diagonal and in a zigzag pattern (rather than an armchair pattern). After prolonged thermal excitation and electron-beam irradiation, all these as-formed vacancies tended to aggregate and line up parallel to the zigzag pattern direction to form extended vacancy lines with a total length reaching hundreds of nanometers or even the micrometer scale. Ab initio calculations were conducted to reveal the vacancy migration pathway, energy landscape, and modifications to the electronic structure of the host BP monolayers (MLs). It was found that the migration of a 5-8-5 DV was accomplished via sequential structural transformations including several transitions and intermediate configurations, such as 5-7-7-5 DVs. The associated migration barriers were determined as 2.1 eV for diagonal migration and 2.6 eV along the zigzag path, respectively. Calculations further confirmed that the presence of vacancy defects induced considerable electronic structure modification of the host ML-BP; for example, the bandgap was reduced from 0.9 eV (for defect-free ML-BP) to 0.7 eV in the presence of vacancy lines with a concentration of 1.2 at.%. The present study expands the current understanding of the formation and dynamic behaviors of primary vacancy defects and illustrates methods available to alter the electronic structures of 2D BP materials. It can further serve as a guideline for the function-oriented design and fabrication of BP-based devices via precisely controlled defect engineering.