BACKGROUND: Malaria remains a serious problem in Colombia. The number of malaria cases is governed by multiple climatic and non-climatic factors. Malaria control policies, and climate controls such as rainfall and temperature variations associated with the El Nino/Southern Oscillation (ENSO), have been associated with malaria case numbers. Using historical climate data and annual malaria case number data from 1960 to 2006, statistical models are developed to isolate the effects of climate in each of Colombia's five contrasting geographical regions. METHODS: Because year to year climate variability associated with ENSO causes interannual variability in malaria case numbers, while changes in population and institutional control policy result in more gradual trends, the chosen predictors in the models are annual indices of the ENSO state (sea surface temperature [SST] in the tropical Pacific Ocean) and time reference indices keyed to two major malaria trends during the study period. Two models were used: a Poisson and a Negative Binomial regression model. Two ENSO indices, two time reference indices, and one dummy variable are chosen as candidate predictors. The analysis was conducted using the five geographical regions to match the similar aggregation used by the National Institute of Health for its official reports. RESULTS: The Negative Binomial regression model is found better suited to the malaria cases in Colombia. Both the trend variables and the ENSO measures are significant predictors of malaria case numbers in Colombia as a whole, and in two of the five regions. A one degree Celsius change in SST (indicating a weak to moderate ENSO event) is seen to translate to an approximate 20% increase in malaria cases, holding other variables constant. CONCLUSION: Regional differentiation in the role of ENSO in understanding changes in Colombia's annual malaria burden during 1960-2006 was found, constituting a new approach to use ENSO as a significant predictor of the malaria cases in Colombia. These results naturally point to additional needed work: (1) refining the regional and seasonal dependence of climate on the ENSO state, and of malaria on the climate variables; (2) incorporating ENSO-related climate variability into dynamic malaria models.