Variable region 1 (V1) of the SPRY domain of TRIM5alpha is a major determinant for species-specific virus restriction in primates. We previously reported that a chimeric TRIM5alpha containing baboon V1 in the background of cynomolgus monkey TRIM5alpha showed potent anti-human immunodeficiency virus type 2 (HIV-2) activity. Since baboons are reportedly sensitive to HIV-2 infection, there was a discrepancy between the ability of baboon TRIM5alpha V1 to restrict HIV-2 and baboon sensitivity to HIV-2. In the study presented here, we examined the roles of V2 and V3 of the baboon TRIM5alpha SPRY domain in its anti-HIV-2 activity. A chimeric TRIM5alpha containing the entire baboon SPRY domain showed weak anti-HIV-2 activity. This attenuation of activity was caused by a single serine-to-proline substitution in baboon TRIM5alpha V2. These findings indicate that the combination of V1 with other variable regions of SPRY is important in anti-HIV-2 activity of primate TRIM5alpha.