Dengue fever, caused by the dengue virus (DENV‐1, −2, −3, and −4), affects millions of people in the tropical and subtropical regions worldwide. Severe dengue is correlated with high viraemia and cytokine storm, such as high levels of transforming growth factor‐β1 (TGF‐β1) in the patient's serum. Here, the TGF‐β1 signaling was investigated in the context of in vitro viral clearance. Macrophages were infected with DENV‐2 at MOI 5 and treated with the TGF‐β receptor 1 and 2 inhibitor, GW788388. TGF‐β1 expression, signal transduction and viral load were evaluated 48 h after DENV‐2 infection by enzyme‐linked immunoassay, immunofluorescence, and RT‐qPCR assays. Total TGF‐β1 levelwas reduced in 15%afterDENV‐2 infection, but the secretion of its biologically active form increased threefold during infection, which was followed by the phosphorylation of Smad2 protein. Phosphorylation of Smad2 was reduced by treatment with GW788388 and it was correlated with reduced cytokine production. Importantly, treatment led to a dose‐dependent reduction in viral load, ranging from 6.6×105 RNA copies/ml in untreated cultures to 2.3 × 103 RNA copies/ml in cultures treated with 2 ng/ml of GW788388. The anti‐TGF‐β1 antibody treatment also induced a significant reduction in viral load to 1.6 × 103 RNA copies/ml. On the other hand, the addition of recombinant TGF‐β1 in infected cultures promoted an increase in viral load to 7.0 × 106 RNA copies/ml. These results support that TGF‐β1 plays a significant role in DENV‐2 replication into macrophages and suggest that targeting TGF‐β1 may represent an alternative therapeutic strategy to be explored in dengue infection.