The antiviral factor CPSF6-358 restricts human immunodeficiency virus type 1 (HIV-1) infection through an interaction with capsid (CA), preventing virus nuclear entry and integration. HIV-1 acquires resistance to CPSF6-358 through an N74D mutation of CA that impairs binding of the antiviral factor. Here we examined the determinants within CPSF6-358 that are necessary for CA-specific interaction. Residues 314 to 322 include amino acids that are essential for CPSF6-358 restriction of HIV-1. Fusion of CPSF6 residues 301 to 358 to rhesus TRIM5α is also sufficient to restrict wild-type but not N74D HIV-1. Restriction is lost if CPSF6 residues in the amino acid 314 to 322 interaction motif are mutated. Examination of the CA targeting motif in CPSF6-358 did not reveal evidence of positive selection. Given the sensitivity of different primate lentiviruses to CPSF6-358 and apparent conservation of this interaction, our data suggest that CPSF6-358-mediated targeting of HIV-1 could provide a broadly effective antiviral strategy.