The present series of experiments were conducted to examine the effect of Japanese Kampo Medicines (JKMs), namely Tokishakuyakusan (TJ23), Keishibukuryogan (TJ25), Shakuyakukansouto (TJ68) and Unkeito (TJ106) on the physiological function of frozen-thawed bovine spermatozoa in in vitro fertilization. In the first experiment (experiment 1), the most effective JKM was determined by adding JKMs to the BSA-free in vitro fertilization medium (m-Hepes-BO) and examining the developmental capacity of oocytes after in vitro fertilization. In the second experiment (experiment 2), a comparison of the effect of the most effective JKM on in vitro fertilization with that of BSA was conducted in the same manner as in experiment 1. The result of experiment 1 showed that Unkeito (TJ106) was the most effective JKM when added to BSA-free m-Hepes-BO medium, although very few blastocysts were obtained. Furthermore, recording the survivability of the frozen-thawed spermatozoa incubated in fertilization medium supplemented with TJ106 and/or BSA showed that the survivability of the spermatozoa was improved by TJ106. In experiment 2, when TJ106 was added to m-Hepes-BO medium together with BSA, the rates of morulae and blastocysts were better than in the case of TJ106 alone. Moreover, the morphological characteristics of oocytes after 6 h of insemination in fertilization medium with BSA and/or TJ106 as the supplements, also indicated that BSA was indispensable in in vitro fertilization of bovine oocytes, althought the physiological functions of the spermatozoa were somewhat improved by JKM in the present work.