Preliminary evidence suggests that nucleotide feeding improves fertility in dairy cows, most likely mediated by an improvement in general and (or) local immunity of the reproductive tract. The objective of this research was to determine the effect of supplementing nucleotides on dairy cow milk production, reproductive performance and immunity. Two hundred and 60 adult (≥2 lactations) Holstein dairy cows (87 d in milk) were divided in 2 groups and fed a standard diet (40% forage, 60% concentrate) without (CONTROL) or with nucleotides supplement (NUCLEOFORCE COWS, Biobierica S.A.) included at 4 g/cow/day during a period of 7 mo (May through December, 2014). Milk production (daily) and composition (monthly) were measured throughout the experimental period, and days from calving to first insemination and calving to pregnancy recorded. All cows were re-vaccinated against clostridia after 6 weeks in treatment. Serum IgG (n = 160) were determine immediately before and 8 weeks after vaccination, and mucus from the vagina (n = 34) was collected on the day of insemination at least 6 weeks after vaccination to measure IgA concentrations. Data were analyzed with PROC GLM of SAS and differences declared at P < 0.05. There were no differences (P > 0.10) in milk production (42.8 kg/d), milk fat content (2.85%) and yield (1.23 kg/d), milk protein content (3.13%) and yield (1.34 kg/d) or somatic cell counts (387.000 counts/mL) between treatments. However, the ratio pre-vaccination vs. post-vaccination serum IgG tended to be higher (P < 0.08) in the nucleotides supplementation (1.04) compared with CONTROL (0.93) treatment. Concentrations of IgA in vaginal mucus were higher (P < 0.05) in nucleotides supplementation compared with CONTROL (2274 vs. 2078 ng/mL) treatment. Results indicate that the supplementation with nucleotides improved the indicators of general and reproductive tract immunity in dairy.