Transvaginal ultrasound-guided Ovum Pick-Up (OPU) is an established technique in other species. Due to several challenges, there are few publications addressing the procedure in sows. An efficient OPU technique may allow for the collection of numerous oocytes from valuable sows for porcine in vitro embryo production, gene editing and cloning programmes, or cryopreservation. We aimed to improve transvaginal OPU and equipment for this technique in sows. In experiment 1, we conducted 13 OPU sessions on three Landrace x Large White hybrid sows under general anaesthesia, while the second experiment explored OPU in non-sedated animals (N = 6) physically restrained in a commercial claw trimming chute. The experiments resulted in 6.6 ± 5.6 (mean ± SD) and 7.7 ± 8.9 recovered cumulus-oocyte complexes per session, respectively. Post-mortem examination of the pelvic and abdominal cavities of the three sows subjected to repeated OPU sessions did not reveal major acute or chronic pathological lesions. The only sow which was inseminated after the experiment delivered 16 liveborn piglets at term. Salivary cortisol levels increased during the procedure in non-sedated and physically restrained sows but returned to baseline 1 h later (n = 5), indicating a short-term stress response. The described OPU technique and equipment have the potential to retrieve considerable numbers of oocytes by repeated procedures on valuable mature sows. Follow-up studies are needed to optimize the efficiency of the aspiration of high-quality oocytes and to describe the developmental competence of these OPU-derived oocytes. It is also essential to further investigate sow welfare during and after the procedure before recommending porcine transvaginal OPU as a sustainable and welfare-friendly procedure.