Leukocyte infiltration into the central nervous system (CNS) is a key pathological feature in multiple sclerosis (MS) and the MS animal model experimental autoimmune encephalomyelitis (EAE). Recently, preventing leukocyte influx into the CNS of MS patients is the main target of MS therapies and insight into cell behaviour in the circulation is needed for further elucidation of such therapies. In this study, we aimed at in vivo visualization of monocytes in a time-dependent manner during EAE. Using intravital two-photon microscopy (IVM), we imaged CX3CR1gfp/gfp mice during EAE, visualizing CX3CR1-GFP+ monocytes and their dynamics in the spinal cord vasculature. Our observations showed that intraluminal crawling of CX3CR1-GFP+ monocytes increased even before the clinical onset of EAE due to immunization of the animals. Furthermore, intraluminal crawling remained elevated during ongoing clinical disease. Besides, the displacement of these cells was larger during the peak of EAE compared to the control animals. In addition, we showed that the enzyme tissue transglutaminase (TG2), which is present in CNS-infiltrated cells in MS patients, is likewise found in CX3CR1-GFP+ monocytes in the spinal cord lesions and at the luminal side of the vasculature during EAE. It might thereby contribute to adhesion and crawling of monocytes, facilitating extravasation into the CNS. Thus, we put forward that interference with monocyte adhesion, by e.g. inhibition of TG2, should be applied at a very early stage of EAE and possibly MS, to effectively combat subsequent pathology.