It has been previously established that dairy cows are immunosuppressed during early lactation. Serotonin has been shown to influence immune function in other species. Therefore, we hypothesized that infusions of a serotonin precursor during lactation would have an impact on immune function in lactating dairy cows. The objective of this study was to evaluate if intravenous (IV) infusions of 5-hydroxytryptophan (5-HTP), in mid- to late-lactation cows will influence immune response. This study utilized 12 multiparous Holstein cows, blocked by parity in a randomized complete block design. The cows were administered either IV 1.5 mg/kg 5-HTP (n = 6) or saline (n = 6) once a day for 3 consecutive days. Baseline blood samples were collected before treatment, as well as 0, 8, and 24 h after the final 5-HTP infusion. Blood samples were analyzed via qPCR to evaluate mRNA expression of general circulating markers of the innate and adaptive immune response. The genes analyzed were: cluster of differentiation 19 (CD19), protein-tyrosine phosphatase receptor type C (CD45), cluster of differentiation 3 gamma (CD3-G), cluster of differentiation 3 delta (CD3-D), cluster of differentiation 3 epsilon (CD3-E), and low affinity immunoglobulin gamma Fc region receptor III-A (CD16-A). There was no difference in gene expression of CD16-A, CD45, CD3-G, CD3-D, or CD3-A expression between treatment groups (P = 0.27, P = 0.22, P = 0.78, P = 0.27, or P = 0.27, respectively). However, CD19, a circulating marker of adaptive immunity, had increased expression in 5-HTP treated cows compared with control cows (P = 0.0025). Milk yield data were collected throughout the treatment, and there was no difference between treatment groups (P = 0.57). Taken together, this data suggests a lack of an innate immune response to infusions of 5-HTP. Further experiments should be conducted to determine the effect of 5-HTP on the adaptive immune response in the dairy cow during lactation.