The endocannabinoid system (ECS) is an endogenous regulatory system involved in a wide range of physiologic and disease processes. Study of ECS regulation provides novel drug targets for disease treatment. Intravital microscopy (IVM), a microscopy-based imaging method that allows the observation of cells and cell-cell interactions within various tissues and organs , has been utilized to study tissues and cells in their physiologic microenvironment. This article reviews the current state of the IVM techniques used in ECS-related inflammation research. IVM with focus on conventional fluorescent microscope has been introduced in investigation of microcirculatory function and the behavior of individual circulating cells in an environment. Experimental setting, tissue protection under physiologic condition, and microscopical observation are described. Using IVM to investigate the effects of immune modulation by cannabinoids is extensively reviewed. The inflammatory disorders include sepsis, arthritis, diabetes, interstitial cystitis, and inflammatory conditions in the central nervous system and eyes. IVM is a critical tool in cannabinoid and immunology research. It has been applied to investigate the role of the ECS in physiologic and disease processes. This review demonstrates that the IVM technique provides a unique means in understanding ECS regulation on immune responses in diseases under their physical conditions, which could not be achieved by other methods.