The aim of study was to investigate the inhibitory effect of methylene blue by determining some kinetic properties of tissues arginase in sheep spleen. The spleen tissues samples used in this study were obtained from Elkas slaughterhouse in Elazıg. Arginase activity was spectrophotometrically measured, using the Thiocemicarbazide diacetylmonoxime urea (TDMU) method. Protein activity was measured with the method of Lowry et al. The study was performed on four groups. In the first group, the samples were supplemented with MnCl2 at the first preincubation and kept at room light. In the second group, the samples were supplemented with methylene blue and MnCl2 at the first preincubation and exposed to a 150 W (watt) light source. In the third group, MnCl2 at first preincubation and methylene blue at the second preinkubation were included in the samples that were then exposed to a 150 W (watt) light source. In the fourth group, after supplementing methylene blue at the first preincubation and MnCl2 at the second preincubation, the samples were exposed to a 150 W light source. The highest decrease in the sheep spleen arginase activity were determined in the fourth group, which were followed by the second group. The least decrease in the enzyme activity was determined in the third group. It was analyzed to have 66, 60 and 72 % of inhibitory decrease in enzyme activity successfully in the room light, dark and 150 w light source. Methylene blue was found to result in a non-competitive inhibitory effect on sheep spleen tissues arginase. We concluded that photoinactivation may be due to a modification in imidazole group of hystidyl residues present in arginase molecules.