Abstract Background Preconception care offers an opportunity to improve women’s health literacy and health behaviours before pregnancy, but scalable real-world models for women in the general population remain incompletely evaluated. Anti-Müllerian hormone (AMH) testing is increasingly familiar to reproductive-aged women, yet its limitations are frequently misunderstood. We evaluated whether a multifaceted preconception care intervention incorporating education, AMH testing and structured post-test counselling was associated with changes in preconception knowledge, AMH literacy and health-behaviour readiness among women aged 25–34 years. Methods This multicentre before-and-after implementation study evaluated a prefecture-wide programme conducted at eight medical institutions in Shiga Prefecture, Japan, between April 2025 and March 2026. Women aged 25–34 years viewed a preconception care educational video, underwent serum AMH testing and received structured counselling when results were disclosed. A study-specific longitudinal questionnaire was administered at baseline, immediately after AMH result disclosure and counselling (approximately 1 month after baseline), and at 3 months after baseline. Paired baseline-to-post-intervention analyses included 208 participants, and 93 completed the 3-month follow-up. Outcomes were a preconception knowledge score (4–12), an eight-item health-behaviour readiness score (8–40), an AMH literacy score (0–3) and concern about future fertility (1–4). Wilcoxon signed-rank and McNemar tests were used for paired comparisons. Results From baseline to immediately after the intervention, knowledge increased from 7.84 ± 1.95 to 10.56 ± 1.69, health-behaviour readiness from 23.92 ± 5.48 to 26.61 ± 4.93, and AMH literacy from 1.95 ± 0.83 to 2.39 ± 0.76 (all P