Background: The development of microsurgical techniques has facilitated the establishment of fully vascularized cardiac transplantation models in small mammals. Anastomotic stenosis and bleeding continue to hamper procedures and limit long-term graft survival. In this study we assess a novel technique to improve outcome after cardiac transplantation in mice. Methods: Our novel technique of murine heterotopic cardiac transplantation consists of three critical steps: (i) a novel procedure for graft harvest; (ii) a modified method for recipient vessel preparation; and (iii) a novel suturing procedure for graft implantation. Importantly, a new knotless suturing technique for end-to-side vascular anastomosis was applied, which allows for adjustment of the anastomosis after transplantation, thus reducing the risk of anastomotic bleeding or stenosis. Results: The recipient survival rate based on this novel technique was between 90% and 98%, depending on physician expertise. Graft implantation time varied between 20 and 25 minutes after the initial 200 training cases. In comparing the standard knot microvascular suturing technique to the new knotless technique carried out by an experienced surgeon, the latter was found to be more efficient by significantly reducing the rate of anastomotic stenosis (0% vs 8% with knot, p < 0.001, n = 200) and anastomotic bleeding (2% vs 7% with knot, p < 0.05, n = 200). Conclusions: This novel technique offers a rapid, easy and effective method for murine heterotopic cardiac transplantation. © 2009 International Society for Heart and Lung Transplantation.