A better knowledge of synovial fluid (SF) ultrastructure is required to further understand normal joint lubrication and metabolism. The aim of the present study was to elucidate SF structural features in healthy joints from three mammalian species of different size compared with features in biomimetic SF. High-resolution structural analysis was performed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and environmental SEM/wet scanning transmission electron microscopy mode complemented by TEM and SEM cryogenic methods. Laser-scanning confocal microscopy (LCM) was used to locate the main components of SF with respect to its ultrastructural organization. The present study showed that the ultrastructure of healthy SF is built from a network of vesicles with a size range from 100 to a few hundred nanometers. A multilayered organization of the vesicle membranes was observed with a thickness of about 5 nm. LCM study of biological SF compared with synthetic SF showed that the microvesicles consist of a lipid-based membrane enveloping a glycoprotein gel. Thus, healthy SF has a discontinuous ultrastructure based on a complex network of microvesicles. This finding offers novel perspectives for the diagnosis and treatment of synovial joint diseases.