Objective To explore the role of pentraxin 3 (PTX3) in transforming growth factor β1 (TGF-β1)-induced fibrosis in orbital fibroblasts (OFs). Methods OFs from thyroid-associated ophthalmopathy (TAO) patients and healthy donors were cultured in vitro. The human recombinant PTX3 (rhPTX3) (1 μg/mL) and human recombinant TGF-β1 (10 ng/mL) were added to the culture medium for stimulation. After stimulation, specific antibody was selected to label the anti-α-smooth muscle actin (α-SMA), and the fibrosis of OFs was observed by fluorescence microscope. The mRNA expression levels of α-SMA, collagen typeⅠα1 (ColIα1) and interleukin 6 (IL-6) were detected by quantitative real-time polymerase chain reaction after the same stimulation. Results TGF-β1 induced the expression of α-SMA protein in the two groups of OFs, and the cells showed fibrotic changes, while the rhPTX3 had no significant inhibition. There was no significant difference in the fibrotic effect of OFs between the patients with TAO and healthy donors. The mRNA expression levels of fibrotic protein (α-SMA, ColIα1, and IL-6) were upregulated by TGF-β1 in OFs. The rhPTX3 had no significant induction for the expression of α-SMA mRNA in OFs, but co-stimulation could significantly enhance the upregulation of α-SMA. The rhPTX could upregulate the mRNA expression of ColIα1 and IL-6. Co-stimulation could enhance the induction of upregulation of IL-6 by TGF-β1, but had no significant effect on the upregulation of ColIα1. Conclusion The rhPTX3 has a potential role in promoting TGF-β1-induced fibrosis in OFs, which indicates that PTX3 may be involved in the later stage of the pathogenesis of diplopia in TAO patients and play a positive role.