Real-time quantitative polymerase chain reaction is a powerful and accurate technique to evaluate gene transcription patterns in different cells under biological and pathological conditions. One of the critical steps in the quantitation of transcription profiles is accurate normalization. In this critical step, use of appropriate stable reference gene(s) would be important. The purpose of this study was to identify the most stable housekeeping genes in equine fibroblast-like synoviocytes (FLS) treated with doxycycline from a panel of candidate genes. The gene transcription levels of six commonly used reference genes (GAPDH, ACTB, PPIA, HPRT1, IPO8, and MRPL19) were determined in normal equine FLS or treated by doxycycline and the combination of both. After applying the BestKeeper, geNorm, and NormFinder programs, to this set of genes, there were differences in the ranked order in the three defined groups. But, overall resultants of all three analysis programs determined that the candidate reference genes could be ranked as HPRT1, ACTB, GAPDH, PPIA, MRPL19, and IPO8. To more accurate normalization of gene quantitative data, using of HPRT1, ACTB, and GAPDH combination as reference gene panels is recommended for equine FLS during normal condition or treated by doxycycline.