Glutamate Carboxypeptidase II (GCPII) is a protease found in many tissues of humans, such as prostate and digestive tract. GCPII is overexpressed in prostate cancer tissue but its role in the cancer remains unknown. One function of GCPII is the transformation of dietary folate to aid in its absorption by cleaving terminal glutamates. Different metabolically relevant forms of folates act as coenzymes in the biosynthesis of DNA, RNA and amino acids. However, the significance of the roles played by GCPII in the broader cellular metabolism is not well known.C. elegans GCPII (cGCPII) is produced as three paralogs: cGCP‐2.1 (gcp‐2.1), cGCP‐2.2 (gcp‐2.2) and cGCP‐2.3 (gcp‐2.3). E. coli OP50 is the primary diet and source of folate for C. elegans and sulfamethoxazole (SMX), a bacterial folate biosynthesis inhibitor, treated E. coli OP50 serves as folate‐deficient diet for this study. Wild type (N2) and three null mutant strains of gcp‐2.1, gcp‐2.2, or gcp‐2.3 genes were fed SMX treated OP50. The longevity of each strain under folate‐deficient conditions was measured. The same strains were fed SMX treated OP50 that was supplemented tetrahydrofolate (THF) or 5‐formyl‐tetrahydrofolate (folinic acid). The brood size of each strain was measured to determine the ability of folate supplementation to restore reproduction observed under folate‐deficient conditions. To identify the folate derivatives present in the OP50 food source, SMX treated and untreated OP50 folate extracts were analyzed by HPLC.Overall, the median longevity increased for all strains that were fed SMX treated OP50 compared to untreated OP50. All paralogous strains show varying longevity, suggesting that cGCPII paralogs may play different roles in longevity. The folate deficient diet significantly reduced brood size in the N2 and the gcp‐2.3 mutant strain compared to the regular diet. The brood size of gcp‐2.1 and gcp‐2.3 mutant strains increased significantly with low level THF supplementation (10 μM) compared to the SMX treated OP50 control group. However, supplementation at a higher concentration (250 μM) caused significant reduction in the brood size of the N2, gcp‐2.1 and gcp‐2.3 strains and a relatively smaller reduction in the gcp‐2.2 strain compared those of SMX treated OP50 controls.After supplementation with folinic acid, the N2 and gcp‐2.2 mutant strains showed significant recovery in reproduction compared to those fed the folate‐deficient diet. The gcp‐2.1 mutant was most affected by folate deficiency, while the gcp‐2.3 mutant was the least affected mutant. HPLC analysis showed tetrahydrofolate, dihydrofolate, and folinic acid were present in the OP50 control extracts. However, none of these derivatives were present in detectable amounts in the SMX treated OP50 extracts. This suggest these folate derivatives may have a contributed to the brood size observations.In conclusion, excessive folate reduces longevity and brood size. However, low levels of folate are required for normal reproduction. The folate‐mediated variation in longevity and brood size, associated with the gcp‐2 paralog present, demonstrate that the gcp‐2 paralogs may have different functional roles in folate metabolism.Support or Funding InformationFunding was received from the Illinois State Academy of Science.