A defect inspection apparatus includes an illumination optical system which sets a transmission illumination region and a reflection illumination region on an inspection target surface of a mask, first and second imaging units having first and second visual fields which are set on the inspection target surface, an imaging optical system that provides images, which are present on the first and second visual fields, on the first and second imaging units, a defect detection unit which detects a defect of the mask on the basis of the images provided on the first and second imaging units, and a control unit which controls a positional relationship between setting positions of the transmission illumination region and the reflection illumination region and setting positions of the first and second visual fields.