We report on the first isolation of dengue virus serotype 4 (DENV-4) in the State of Mato Grosso do Sul, Brazil, in February, 2012. The cases were isolated in the city of Campo Grande, the state capital, and presented the classic signs and symptoms of dengue fever. DENV-4 was primarily identified through viral isolation in C6/36 clone lineage of Aedes albopictus cells; followed by indirect immunofluorescence, using type-specific monoclonal antibodies. The results were subsequently confirmed by Nested RT-PCR tests. The first description of the introduction of DENV-4 in a state whose population is susceptible to this serotype and the circulation of three other serotypes in the area is cause for concern due to the increased possibility of severe and lethal cases of the disease, and of huge epidemics.