Mycobacterium tuberculosis (Mtb) infects different populations of macrophages. Alveolar macrophages (AMs) are initially infected, and their response may contribute to controlling Mtb infection and dissemination. However, Mtb infection may disseminate to other tissues, infecting a wide variety of macrophages. Given the difficulty in obtaining AMs, monocyte-derived macrophages (MDMs) are used to model macrophage–mycobacteria interactions in humans. However, the response of other tissue macrophages to Mtb infection has been poorly explored. We have compared MDMs, AMs and splenic human macrophages (SMs) for their in vitro capacity to control Mtb growth, cytokine production, and induction of cell death in response to Mtb H37Rv, and the Colombian isolate UT205, and to the virulence factor ESAT-6. Significant differences in the magnitude of cell death and cytokine production depending mainly on the Mtb strain were observed; however, no major differences in the mycobacteriostatic/mycobacteriocidal activity were detected among the macrophage populations. Infection with the clinical isolate UT205 was associated with an increased cell death with membrane damage, particularly in IFNγ-treated SMs and H37Rv induced a higher production of cytokines compared to UT205. These results are concordant with the interpretation of a differential response to Mtb infection mainly depending upon the strain of Mtb.