An adequate and clean artificial insemination (AI) technique is recommended to maximize reproductive outcomes in dairy cattle. The objective of this study was to evaluate the effectiveness of using protective sheaths (PS; Continental Plastic Inc., Delavan, WI, USA) to minimize contamination of the AI catheter (AIC) on pregnancies per AI (P/AI) in lactating dairy cattle. A previous study reported no improvement on cattle fertility when using PS during first service AI (King et al. 1984 Can. Vet. J. 25, 327). Lactating cows housed in free-stall barns on a commercial dairy farm with a rolling herd milk production average of 10.140 kg were presynchronized with 2 injections of PGF_2α (25 mg; Lutalyse, Pharmacia & Upjohn, Kalamazoo, MI, USA) given 14 days apart (starting at 26 ± 3 d postpartum) followed by Ovsynch [OV; GnRH-7 d-PGF_2α-56 h-GnRH-16 h-timed-AI(TAI)] 12 days later. Cows presenting signs of standing heat any time during the protocol received AI, whereas the remaining animals were subjected to TAI16 h after second OV GnRH (100 μg; Cystorelin, Merial, Duluth, GA, USA). At the moment of AI (one AI technician), 996 services from lactating dairy cows were randomly assigned to 1 of the 2 groups; with (TRT, n = 487) or without (CON, n = 509) the use of PS. In the TRT group, the AIC protected with a PS was introduced into the vagina; once in the cranial portion of the vagina adjacent to the cervical os, the PS was pulled back and only the AIC was manipulated through the cervix into the uterine body for semen deposition. In the CON group, cows received AI without the PS. Additionally, sterile cotton swab (BD, Franklin Lakes, NJ, USA) samples were taken from AIC (n = 51) after AI from both treatment groups. Pregnancy diagnosis was determined by ultrasonography 42 ± 3 d after AI. Data analyses were performed using GLIMMIX (P/AI) and FREQ (culture) procedures of SAS (SAS Institute Inc., Cary, NC, USA). Cultured swab samples revealed that the use of PS was effective in minimizing contamination of the AIC (bacterial growth on AIC; TRT = 57.7% v. CON = 100%; P < 0.0002). Overall, the proportion of cows pregnant (all services) was greater (P = 0.03) for cows in TRT (42.7 ± 2.2%) compared with CON group (36.1 ± 2.1%). For first services postpartum, P/AI did not differ (P = 0.87) between CON (43.01 ± 4.4%) and TRT (43.8 ± 4.6%) groups. However, P/AI for second or greater services (≥2S) were greater (P = 0.007) in TRT (43.8 ± 2.9%) than in CON cows (32.3 ± 2.6%). Results from this study suggested that the use of PS during AI improved P/AI for ≥2S in lactating dairy cows. Performing a clean AI technique through the use of PS may be a cost-effective strategy to improve reproductive outcomes in dairy cattle. Further investigation is needed under various reproductive management conditions to confirm and determine the underlying mechanisms for these findings.