The main objective of the study conducted here was to estimate the concentration of 2,3-Bisphosphoglycerate (2,3-BPG), 1,3-Bisphosphoglycerate (1,3-BPG), bisphospho-glycerate mutase (BPGM) and 3-phosphoglycerate (3PG) in cattle clinically diagnosed with acute ruminal acidosis. A secondary objective was to examine the physical and chemical characteristics of the ruminal fluid in affected cattle. A total of 20 cattle clinically diagnosed with acute ruminal acidosis and eight clinically normal cattle were included in this study. The results showed that decrease of ruminal pH changed the ruminal fluid colour, odour and consistency, as well as decreased the sedimentation time, increased the methylene blue reduction time, and decreased ruminal microflora motility. The study indicated that the concentration of 2,3-BPG, BPGM and BPGP decreased with the decrease of ruminal pH, while 3-PG concentration was not affected with the decrease of ruminal pH. In conclusion, 2,3-BPG could play a role in the pathogenesis of ruminal acidosis, and thus, the intravenous administration of sodium bicarbonate is important, particularly in severe cases, to correct any systemic acidosis that can decrease 2,3-BPG concentration and results in tissue hypoxia.