Vincristine (VCR) is widely used in cancer therapies, although its benefits on cardiac fibrosis remain unknown. Here, we investigated VCR’s efficacy on cardiac fibrosis and elucidated the underlying mechanism of action. Network pharmacology was employed to predict the mechanism of VCR action on cardiac fibrosis. We induced cardiac fibrosis in adult male Sprague–Dawley (SD) rats via isoproterenol (ISO) injection, followed by treatment with VCR or vehicle. After 10 days of treatment, VCR-treated rats exhibited a significantly lower heart/body weight ratio relative to those treated with the vehicle. Moreover, cardiac fibrosis was alleviated in VCR-treated rats relative to vehicle-treated rats. The results revealed the down-regulation of mature caspase-1, interleukin (IL)-1β, and IL-18 in VCR-treated rats relative to vehicle-treated rats. We also observed less colocalization between the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) and apoptosis-associated speck-like protein containing a CARD (ASC) in VCR-treated rats compared with vehicle-treated rats. We then cultured neonatal rat cardiac fibroblasts (NRCFs) and exposed them to lipopolysaccharide (LPS) and adenosine triphosphate (ATP) in the presence or absence of VCR. The results indicated that VCR mediated the down-regulation of caspase-1, IL-1β, and IL-18 and the colocalization of NLRP3 and ASC in LPS+ATP-stimulated cardiac fibroblasts (CFs). We found evidence that VCR attenuates cardiac fibrosis by directly suppressing the activation of the NLRP3 inflammasome. These findings provide novel insights into VCR’s mechanism of action in alleviating cardiac fibrosis.