Viral corneal infection is a common cause of visual impairment and blindness. Polyinosinic‑polycytidylic acid, or poly(I:C), is similar to viral double‑stranded RNA in structure and has been implicated in the release of a variety of cytokines, chemokines and matrix metalloproteinases (MMPs) by corneal fibroblasts. Sulforaphane (SFN) is an isothiocyanate compound found in cruciferous vegetables. The present study investigated the potential effect of SFN on the poly(I:C)‑stimulated release of cytokines, chemokines and MMPs in human corneal fibroblasts (HCFs). ELISA showed that SFN was associated with a time‑ and dose‑dependent reduction in poly(I:C)‑stimulated production of interleukin (IL)‑8, chemoattractant protein‑1, IL‑6, MMP‑1 and MMP‑3 by HCFs. Western blot analysis indicated that SFN suppressed the function of poly(I:C) by modulating mitogen‑activated protein kinases (MAPKs), including p38 and extracellular signal‑regulated kinase (ERK), activator protein‑1 (AP‑1) component c‑Jun and the kinase, Akt, and the phosphorylation and degradation of the nuclear factor (NF)‑κB inhibitor IκB‑α. Immunofluorescence analysis revealed that SFN attenuated the production of poly(I:C)‑induced nuclear translocation of the NF‑κB p65 subunit. Reverse transcription‑quantitative PCR analysis revealed that SFN prevented the poly(I:C)‑induced upregulation of Toll‑like receptor 3 (TLR3) mRNA expression in HCFs. No significant cytotoxic effect of SFN on HCFs was observed. In summary, SFN attenuated the poly(I:C)‑induced production of proinflammatory chemokines, cytokines and MMPs by HCFs, by inhibiting TLR3, MAPK (p38 and ERK), AP‑1, Akt and NF‑κB signaling. SFN may therefore be a potential novel treatment for viral corneal infection by limiting immune cell infiltration.