FOXO3 is a member of the FOXO (forkhead box class O) transcription factor family and has roles in cell cycle control, apoptosis, neural and hematopoietic cell differentiation and DNA repair among other functions. Several human studies provide evidence for an association between aging, longevity and variation in FOXO3. Recently variation has been identified in exon 2 of ovine FOXO3. This exon encodes the C-terminus of the DNA-binding domain and a transcription activation domain that is the key regulator of transcriptional activity in target genes. The association of genetic variation in exon 2 with lifespan was investigated in 1,732 New Zealand (NZ) sheep. Of the seven haplotypes detected, the presence of the D haplotype, which codes for an amino acid substitution (a conserved methionine residue is replaced by a valine) at residue 407, is associated with a decrease of 0.39 years in mean age (P = 0.034). Significant differences in mean age were also detected between genotypes containing D (AD: 4.7 ± 0.21; BD: 4.7 ± 0.25) and genotypes that did not contain D (AA: 5.1 ± 0.14; AB: 4.9 ± 0.18; BB: 5.3 ± 0.25). Other genotypes were rare in the sheep investigated and were not analyzed. This suggests that genetic variation in ovine FOXO3 influences the lifespan of sheep either directly or indirectly by impacting on factors that lead to reduced productivity and increased likelihood of culling.