Pretarsal orbicularis oculi muscle (POOM) is an important structure of eyelid movement in human. The aim of this study was to investigate fiber histomorphology and myosin heavy chain (MyHC) isoform composition of adult POOM, and to clarify their age-related changes. Eyelid specimens from 58 subjects (age range, 21 to 91 years) were collected during upper blepharoplasty procedures. Serial cross sections of POOM were ATPase-stained and examined under miscroscope. Quantitative measures of muscle fiber size and fiber type distribution were obtained in 35 subjects with adequate fiber cross sections. Relative MyHC isoform contents of POOM were retrieved by gel electrophoresis in all 58 subjects. Examination of the histochemical staining revealed an abundance of type II fiber ( >85%) in human POOM, with more type IIX than IIA fibers. Decreased mean area of all fibers and type IIA fibers were noted in the old group when compared to the young. As for MyHC analysis, the relative content of MyHC isoforms exhibited an order of IIX > IIA > I, and the relative MyHC IIA content showed a negative correlation with age. Comparing with previous studies of limb or masticatory muscles, adult POOM exhibits a unique fiber and MyHC composition, as well as a different aging pattern.