The dairy industry relies on having appropriate reproductive performance to ensure milk production and replacement heifers. Intrauterine infusion of dextrose (i.e., 50% dilution) has been proven to cure clinical endometritis and increase pregnancy rates in dairy cows. The objectives of the present study were to (1) assess the effects of the intrauterine infusion of a 50% dextrose solution on non-pregnant cows, and (2) to estimate the prevalence of subclinical endometritis (SCE) at the time of non-pregnancy diagnosis in lactating dairy cows. Holstein lactating cows (n = 711) from 3 commercial dairy herds were enrolled at non-pregnancy diagnosis, blocked by parity (primiparous and multiparous) and repeat breeder (RB) status (RB ≥ 3 services; noRB < 3 services), and were randomly allocated to receive the intrauterine infusion with a dextrose solution (DEX; n = 356) or remain as controls cows (CON; n = 355). In addition, SCE was assessed on a subset of cows (DEX = 100; CON = 100). Immediately after non-pregnancy diagnosis, DEX cows were infused with 120 mL of a 50% dextrose solution using individually wrapped, single-use sterile infusion pipettes; while CON cows received no treatment. In animals were SCE was evaluated, samples for assessment of polymorphonuclear cells were collected from the uterine body using the cytobrush technique immediately before treatment administration. All cows followed the established on-farm reproductive program for subsequent AI. Pregnancy (PAI) was determined by the herd veterinarian at 40 ± 5 d post-AI. No differences (P ≥ 0.05) in the prevalence of SCE were observed between DEX and CON cows. Overall, no difference (P ≥ 0.05) in PAI were observed between cows in the DEX (31.3 ± 2.5%) and CON (26.6 ± 2.3%) groups. However, analysis by RB status showed that noRB cows within the DEX group had higher (P < 0.05) PAI when compared with cows in the CON group (36.6 ± 3.6% and 26.9 ± 3.4% respectively). Interestingly, PAI in RB cows treated with DEX (24.8 ± 3.4%) was not different (P ≥ 0.05) from CON (26.1 ± 3.3%). In conclusion, the intrauterine infusion with 120 mL of a 50% dextrose solution may alter the uterine environment early in lactation as reflected by the improved PAI in noRB dairy cows.