The objective of this study was to investigate the prognostic value of single and repeated measurements of blood l-lactate (Lac) and ionised calcium (iCa) concentrations, packed cell volume (PCV) and plasma total protein (TP) concentration in horses with acute colitis. A total of 66 adult horses admitted with acute colitis (<24 h) to a referral hospital in the 2002–2011 period were included. The prognostic value of Lac, iCa, PCV and TP recorded at admission and 6 h post admission was analysed with univariate analysis, logistic regression, classification and regression trees, as well as random forest analysis. Ponies and Icelandic horses made up 59% of the population, whilst the remaining 41% were horses. Blood lactate concentration at admission was the only individual parameter significantly associated with probability of survival to discharge (P < 0.001). In a training sample, a Lac cut-off value of 7 mmol/L had a sensitivity of 0.66 and a specificity of 0.92 in predicting survival. In independent test data, the sensitivity was 0.69 and the specificity was 0.76. At the observed survival rate (38%), the optimal decision tree identified horses as non-survivors when the Lac at admission was ≥4.3 mmol/L and the Lac 6 h post admission stayed at >2 mmol/L (sensitivity, 0.72; specificity, 0.8). In conclusion, blood lactate concentration measured at admission and repeated 6 h later aided the prognostic evaluation of horses with acute colitis in this population with a very high mortality rate. This should allow clinicians to give a more reliable prognosis for the horse.