Hypocalcemia affects 50% of dairy cows. Our lab has previously demonstrated that infusions of the serotonin precursor 5-hydroxy-l-tryptophan (5-HTP) increases circulating calcium levels in the transition cow. It is unknown whether feeding a negative DCAD diet alters the relationship between 5-HTP and hypocalcemia. The main objective of this study was to determine whether feeding a negative DCAD diet before calving in conjunction with a 5-HTP treatment could further diminish the magnitude of hypocalcemia at the time of calving. We utilized a randomized complete block design with a 2 × 2 factorial arrangement. 32 multiparous Holstein cows were fed either a positive (+130 mEq/kg) or negative (−130 mEq/kg) DCAD diet 21 d before calving and were treated daily with saline or 5-HTP (1 mg/kg) IV starting 7 d before estimated calving date. Cows were blocked by parity and were randomly assigned to one of 4 treatment groups: positive DCAD plus saline (+DCAD/CON), positive DCAD diet plus 5-HTP (+DCAD/5-HTP), negative DCAD plus saline (-DCAD/CON), and negative DCAD plus 5-HTP (-DCAD/5-HTP), resulting in an n = 8 per group. Total calcium (tCa), ionized calcium (iCa), and feed intake (DMI) were recorded. iCa was significantly elevated pre-calving (P = 0.02) in the -DCAD/5-HTP group compared with the other treatment groups as well as on d 0 and 1 after calving (P < 0.001). While differences in tCa were not significant across the pre or post-calving periods, tCa was numerically higher on d 0 and significantly higher on d 1 in the -DCAD/5-HTP (P < 0.05) cows compared with all other groups. While there was a DCAD by treatment interaction on DMI (P = 0.03) pre-calving, post-calving DMI differences were not significant. These findings demonstrate that feeding a -DCAD diet in conjunction with 5-HTP pre-calving can increase post-calving circulating Ca concentrations and therefore diminish the magnitude of hypocalcemia at the time of calving.