RRx-001 is a pleiotropic anticancer agent in phase III clinical trials, which polarizes tumor-associated macrophages from a low phagocytic M2 phenotype to a high phagocytic M1 phenotype. One of the ways in which tumors promote M2 polarization and evade macrophage-mediated destruction is through upregulation of CD47 expression. As a myeloid-specific immune checkpoint, CD47 interacts with signal-regulatory protein alpha (SIRPα) on macrophages and monocytes to prevent phagocytosis. Herein, the effect of RRx-001 on CD47 and SIRPα expression was evaluated as well as its activity in vivo in macrophage-depleted tumors. In vitro, RRx-001 was found to decrease the expression levels of CD47 and SIRPα on tumor cells and monocytes/macrophages, respectively, reducing the phagocytosis inhibitory function of the CD47/SIRPα interaction. In vivo, macrophage depletion by clodronate in an A549 xenograft-bearing mouse model attenuated the ability of RRx-001 to suppress tumor growth, which suggests that the presence of infiltrated macrophages in the tumor microenvironment is a sine qua non condition for the antitumor activity of RRx-001. Furthermore, these in vitro effects translate into significant antitumor activity in mouse models of lung cancer. Importantly, unlike anti-CD47 antibodies, RRx-001, which has been evaluated in close to 300 patients in 9 clinical trials, is not associated with any hematologic toxicities. On the basis of demonstrated antitumor activity and minimal toxicity in phase II clinical trials, RRx-001 has received clearance from the FDA and the EMA for phase III, multicenter studies in subjects with relapsed/refractory solid tumors.