Corneal immune privilege is integral in maintaining the clear avascular window to the foreign world. The presence of distinct populations of corneal leukocytes (CLs) in the normal cornea has been firmly established. However, their precise function and kinetics remain, as of yet, unclear. Through intravital multiphoton microscopy (IV-MPM), allowing the means to accumulate critical spatial and temporal cellular information, we provide details for long-term investigation of CL morphology and kinetics under steady state and following inflammation. Significant alterations in size and morphology of corneal CD11c dendritic cells (DCs) were noted following acute sterile inflammation, including cell volume (4364.4 ± 489.6 vs. 1787.6 ± 111.0 μm, P < 0.001) and sphericity (0.82 ± 0.01 vs. 0.42 ± 0.02, P < 0.001) compared with steady state. Furthermore, IV-MPM analyses revealed alterations in both the CD11c DC and major histocompatibility complex class II (MHC)-II mature antigen-presenting cell population kinetics during inflammation, including track displacement length (CD11c: 16.57 ± 1.41 vs. 4.64 ± 0.56 μm, P < 0.001; MHC-II: 9.03 ± 0.37 vs. 4.09 ± 0.39, P < 0.001) and velocity (CD11c: 1.91 ± 0.07 μm/min vs. 1.73 ± 0.1302 μm/min; MHC-II: 2.97 ± 0.07 vs. 1.62 ± 0.08, P < 0.001) compared with steady state. Our results reveal in vivo evidence of sessile CL populations exhibiting dendritic morphology under steady state and increased velocity of spherical leukocytes following inflammation. IV-MPM represents a powerful tool to study leukocytes in corneal diseases in context.-Seyed-Razavi, Y., Lopez, M. J., Mantopoulos, D., Zheng, L., Massberg, S., Sendra, V. G., Harris, D. L., Hamrah, P. Kinetics of corneal leukocytes by intravital multiphoton microscopy.