Trypanosoma theileri is considered a non- or low-pathogenic trypanosome that generally causes latent infection in apparently healthy cattle; however, T. theileri propagates in the bloodstream and may cause clinical disease in pregnant animals or co-infection with bovine leukemia virus or Theileria orientalis. In the current study, a monthly survey of T. theileri infection over one year was carried out in a research dairy farm in Hokkaido, Japan to determine the 1) seasonal variations in the prevalence, 2) genetic characterization of T. theileri, and 3) associations of milk and blood parameters in dairy cattle with T. theileri infection, including data of metabolic profile tests and dairy herd performance tests, using linear mixed models. We found that 1) the prevalence of T. theileri infection was significantly higher in summer and winter than in other seasons; 2) T. theileri possibly showed genetic diversity in Eastern Hokkaido; and 3) T. theileri infection was associated with significantly lower levels of blood urea nitrogen, milk protein, and solids-not-fat, which are caused by a low rumen fermentation level. This is the first study to report the negative impact of T. theileri infection in dairy cattle, and our study indicates that control of T. theileri infection can improve the productivity of dairy cattle.