Purpose: To investigate the relationship between Acanthamoeba cysts and inflammatory cells in Acanthamoeba keratitis (AK) by in vivo confocal microscopy (IVCM). Methods: A case-control study included 30 patients with AK and 20 normal controls. The severity of the AK was divided into mild, moderate, and severe. The central cornea and four standard quadrants of the peripheral cornea were imaged by IVCM. The cyst infiltration and dendritic cell (DC) density and maturity (size, length, field, and number of dendrites) were quantified. The relationship between clinical severity, cyst density, and DC alterations was characterized by Spearman correlation analysis. Results: The maximum cyst density in the mild, moderate, and severe groups was 31.3 cysts/mm(2) (17.2-32.8), 62.5 cysts/mm(2) (59.3-103.1), and 162.5 cysts/mm2 (65.6-215.6), respectively. Compared to normal participants, a significant increase in the mean corneal DC density was detected in patients with AK (290.2 +/- 97.0 vs. 25.3 +/- 8.3 cells/mm(2); P < 0.001). Patients with AK presented an increase in median DC size (178.3 vs. 63.6 mu m(2)/cell, P < 0.001), median DC field (518.1 vs. 256.6 mu m(2)/cell, P = 0.008), and median DC dendrite length (42.3 vs. 14.7 mu m/cell, P < 0.001). Increased AK severity was correlated with an increase in cyst density, DC size, and dendrite length (all P < 0.05). An increase in cyst density was significantly correlated with an increase in DC density (beta = 0.484, P = 0.026) and DC size (beta= 0.557, P = 0.009). Conclusions: Cyst density and depth of infiltration as well as maturity of the surrounding DC increased significantly with the severity of AK. Translational relevance: Quantitative analysis of cyst density and DC maturity may provide a new method of evaluating the severity of AK.