Gene expression of bovine trophoblast cell line (BT-1) was analyzed with a custom utero-placenta complementary DNA microarray. Expression comparison with in vivo tissues of trophoblast derivation was performed to investigate characteristics of the expression in BT-1. BT-1 is a cell line established without feeder cells using trophoblast cells that are separated from bovine blastocysts. The bovine in vivo tissues of the trophoblast derivation were collected on day 17 through 56 of gestation. Of 1,773 genes, 933 exhibited an expression difference exceeding two-fold between BT-1 and in vivo tissues. These genes were analyzed by the k-means clustering method and were distributed into six clusters. Some genes, such as placental lactogen, interferon-τ and pregnancy-associated glycoproteins, all known as trophoblast-specific genes, were detected in BT-1 as well as in vivo tissues throughout the experiment period. These trophoblast-specific genes and octamer-binding transcription factor-4, known as a marker for undifferentiation of cells in mice, were detected by RT-PCR in both BT-1 and in vivo trophoblast tissues. The overall gene expression profile in BT-1 suggests that this cell line contains trophoblast-specific characteristics and is similar to trophoblast cells around the implantation period.