Purpose: To characterize the microscopic anatomy of the lacrimal punctum and canaliculi in relation to the tarsal plate, muscle of Riolan, and Horner muscle; and to report a novel technique to excise the horizontal canaliculus in severe dry eye patients. Design: Observational anatomic study and a retrospective case series. Methods: The microscopic anatomy was studied in 86 eyelids of 25 cadavers (age range: 4596 years, mean: 79.5 years). Surgery was performed on 18 canaliculi of 7 patients with dry eyes (age range: 3769 years, mean: 59.9 years). In the microscopic study, 32 eyelids were incised sagittally, 38 eyelids were incised horizontally (1 mm from the eyelid margin), and 16 eyelids were incised parallel to the tarsal plate. All specimens were stained with Masson trichrome. In the surgical group, probe-guided horizontal canalicular excision with incision of the Horner muscle to the lateral edge of the lacrimal caruncle was performed. Both canalicular stumps were cauterized. Results: In the microscopic anatomic study, the punctum and the vertical canaliculus were part of the tarsal plate with the muscle of Riolan, whereas the horizontal canaliculus was surrounded by the Horner muscle. In the surgical group, all the operated canaliculi were completely occluded without recanalization 12 months postoperatively. No complications were recorded. Conclusions: Based on microscopic anatomic findings that the lacrimal punctum and the vertical canaliculus are part of the tarsal plate, and that the horizontal canaliculus is surrounded by the Horner muscle, excision of the horizontal canaliculus may be an effective technique to treat patients with severe dry eyes.