The separate distolingual (DL) roots of three-rooted mandibular first molars are thought to be too difficult for performing apical surgery. This article represents microsurgical treatment of a three-rooted mandibular first molar with a separate DL root. The procedure includes incision and flap retraction, osteotomy, apicoectomy, retropreparation and retrofilling of the root canal, using micro instruments, ultrasonic retrotips and mineral trioxide aggregate (MTA) under a dental operating microscope. Two mm in length of apical root resection, 2 mm in depth of root canal retropreparation with a personalised ultrasonic retrotip, and 2 mm in length of retrofilling with MTA are the key points for accomplishment of apical surgery on separate DL roots. The case was followed up for 15 months after surgery. Clinical and radiographic examinations revealed complete healing of periapical tissue. Separate DL roots of three-rooted mandibular first molars can be treated by endodontic microsurgery with modifications from standard protocol.