The high glycine/tyrosine type II keratin protein 6.1 (KAP6.1) is a member of the keratin-associated protein family, which is restricted to cells in hair follicles and is associated with fiber diameter and fiber curvature in Merino sheep. In this study, we obtained a series of progressive 5'-deletion fragments of the KAP6.1 gene promoter from ovine genomic DNA. The KAP6.1 5'-upstream region was fused to luciferase and transfected into sheep fetal fibroblast cells (sFFCs). We demonstrated that the sequence from -1,523 to -1 bp (taking the A of the initiator methionine ATG as the +1 nt position) gave rise to a higher luciferase activity comparing to the published region from -1,042 to -1 bp. Whereas, decreased transcriptional activity of the KAP6.1 promoter was observed when the sequence extended to -3,699 bp. To identify the DNA elements that are important for transcriptional activity, we performed structural analysis and electrophoretic mobility shift assay (EMSA). Structural analysis of the KAP6.1 promoter showed that transcription factors NF-kappa-B, AP-1, and C/EBP-alpha synergistically activate KAP6.1 promoter, while POU2F1 might function as a strong negative regulator. The EMSA showed that NF-kappa-B element bound to the nuclear protein extracted from sFFCs. We conclude that NF-kappa-B binding site is an enhancer element of KAP6.1 promoter in vitro. The results are potentially useful for elucidating the regulator mechanisms of KAP6.1.