Cardiac fibroblasts (CFBs) play pivotal roles in myocardial fibrosis, which is the leading cause of arrhythmia. This study was aimed to investigate the modulation of microRNA (miR)-33a on proliferation, apoptosis and fibrosis of human CFBs. CFBs were respectively transfected with miR-control, miR-33a mimic or miR-33a inhibitor, followed by induction of transforming growth factor-β (TGF-β). Non-treated CFBs acted as control. Cell viability, apoptosis, and fibrosis which reflected by expressions of Col-I, Col-III and α-smooth muscle actin (α-SMA) were evaluated by CCK-8 assay, flow cytometry, qRT-PCR and Western blot analysis. Finally, key kinases involved in the TGF-β/Smad pathway were evaluated by Western blot analysis. TGF-β enhanced CFB viability, and expression levels of Col-I, Col-III and α-SMA in CFBs (P < 0.01 or P < 0.001). The increased CFB proliferation, and upregulation of Col-I, Col-III and α-SMA were all further enhanced by miR-33a mimic (P < 0.05 or P < 0.001), whereas reversed by miR-33a inhibitor (P < 0.05, P < 0.01 or P < 0.001). The CFB apoptosis was remarkably promoted by miR-33a inhibitor (P < 0.001). Results of signaling pathway showed that phosphorylated levels of Smad-2 and Smad-3 were both upregulated by TGF-β (P < 0.001). The upregulated phosphorylations were further improved by miR-33 mimic (P < 0.05) while reversed by miR-33a inhibitor (P < 0.05 or P < 0.001). miR-33a deficiency inhibits proliferation and fibrosis of CFBs while promotes CFB apoptosis by inactivation of TGF-β/Smad pathway.