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Herein, we aimed to evaluate cultures of femoral chondrocytes from offspring of rats with intrauterine growth restriction (IUGR) induced by maternal hyperthyroidism. Fourteen adult female Wistar rats were divided into two groups, a control group and a group treated with daily L-thyroxine administration using an orogastric tube (50 µg/animal/day) during pregnancy. Three days after birth, the offspring were euthanized for chondrocyte extraction. At 7, 14, and 21 days, viability and alkaline-phosphatase (ALP) activity were assessed using the MTT assay and BCIP/NBT method, respectively, in a 2D culture. Pellets (3D cultures) were stained with periodic acid Schiff (PAS) to assess the morphology and percentage of PAS+ areas. The gene transcripts for Col2, Col10, Acan, Sox9, and Runx2 were evaluated by qRT-PCR. The MTT and ALP-assay results showed no significant differences between the groups. Maternal hyperthyroidism did not alter the chondrocyte morphology, but significantly reduced the percentage of PAS+ areas, decreased the expression of the gene transcripts of Col2 and Acan, and increased Sox9 expression. Maternal hyperthyroidism in rats alters the composition and gene expression of the matrix produced by chondrocytes from offspring with IUGR. This may be one of the mechanisms through which excess maternal thyroid hormones reduce offspring bone growth.