The monoclonal antibody (Mab), which recognized CD molecule on lymphocytes, is useful tool for monitoring the immunological status by Flow cytometory (FCM) analysis. CD4 is a glycoprotein found on surface of helper T cells, monocyte, macrophage, and dendritic cells. In this study, we found Japanese black (JB) calves that did not react with one of CD4 specific Mab. To identify the low-reactive calves, we examined for 21 JB calves (1-12 month of age) bred at the same farm using two different bovine CD4 Mabs (clone #CC8 and #CACT138A). FCM analysis showed that the calves were classified into two groups by reactivity against Mabs, i.e. double positive (DP) group react to both clone of Mabs, or single positive (SP) group react to only #CACT138A. A total of seven calves did not react to #CC8, but these calves showed normal reaction to another Mab #CACT138A. The sequencing analysis of these CD4 genes disclosed four nucleotide substitutions (G918T, A930C, G970A, G1074A) in the coding region of SP group as compare to DP group. The three of four these mutations were associated with amino acid substitution (Q306H, K310N, A324T). The substitution at A324T was included in the D4 domain of CD4 gene. Homology modeling based on their amino acids sequence revealed that the surface structure of this part was greatly changed compare to the structures of DP group. Therefore the epitope of CD4 Mab have been changed by this genetic mutation and it might follow low-reactive calves (SP group) against #CC8 Mab. In conclusion, this is the first study to identify CD4 variant in Japanese black cattle and we confirmed that the variants did not affect lymphocyte function such as mitogen stimulation and LPS induced cytokine gene expressions.