A hypothesis was tested that long-term administration of thyroxine (T4) in broiler breeder hens would affect fertility, sperm penetration rate, and the duration of fertility. Relative abundance of oviductal TGF-β4 and HSP70 mRNA was determined to ascertain whether T4 treatment affected these genes, and modulated the sustained storage of spermatozoa within the uterovaginal sperm storage tubules of hens. A total of 70, 47-week-old Cobb 500 breeder hens was randomly allotted to two treatment groups (T4 treatment (ET) and control). The T4 was orally administered to the ET group (0.3 mg T4/bird/day) for 100 consecutive days; whereas the control group was not administered T4 during the experimental period. Breeder hens were artificially inseminated to evaluate specific reproductive variables. On the last day of the treatment period two hens /replicate were randomly killed to estimate oviductal gene expression. The T4 treatment resulted in an increase in plasma concentration of T4; however, the T3 concentration was not affected. The long term administration of T4 had no effect on fertility; however, it resulted in a decreased sperm penetration rate and decreased the duration of fertility compared with the control group. The relative abundance of TGF-β4 and HSP70 mRNA in the SST was not influenced by T4 supplementation. The correlation coefficients between fertility and sperm penetration rate with relative abundance of TGF-β4 and HSP70 were not significant. Overall, among the diverse reproductive variable assessed in the current study, the sperm penetration rate and the duration of fertility were most responsive to long-term treatment with T4. Copyright © 2018 Elsevier B.V. All rights reserved.