Malignancy is a major cause of late post-heart transplantation (HT) mortality. Sirolimus (SRL) exerts antiproliferative properties and its long-term use in HT as primary immunosuppression (IS) is associated with decreased mortality risk that is not fully explained by attenuation of cardiac allograft vasculopathy progression. This study sought to examine whether conversion from calcineurin inhibitor (CNI)-based to SRL-based IS was associated with decreased risk of malignancy post-HT. Overall, 523 patients underwent HT between 1994 and 2016 at a single institution. The main outcomes included incidence of overall de novo malignancies (excluding non-melanoma skin cancers [NMSCs]), post-transplantation lymphoproliferative disorders (PTLD), and first and subsequent primary occurrences of NMSC post-HT. The study identified 307 patients on SRL-based and 216 on CNI-based maintenance IS. Over a median follow-up of 10 years after HT, overall de novo malignancies (non-NMSC) occurred in 31% of CNI patients and in 13% of SRL patients (adjusted hazard ratio [HR]: 0.34; 95% confidence interval [CI]: 0.18 to 0.62; p < 0.001). The incidence of the first NMSC was similar in the SRL and CNI groups (HR: 0.92; 95% CI: 0.66 to 1.28; p = 0.62). However, conversion to SRL was significantly associated with a decreased risk of subsequent primary occurrences of NMSC compared with that of CNI (adjusted HR: 0.44; 95% CI: 0.28 to 0.69; p < 0.001). The adjusted PTLD risk was significantly decreased in the SRL group (HR: 0.13; 95% CI: 0.03 to 0.59; p = 0.009). Late survival post-HT was markedly decreased in patients who developed non-NMSC, PTLD, or non-PTLD compared with patients who did not develop these malignancies, whereas NMSC had no significant effect on survival. Conversion to SRL was associated with a decreased risk of all de novo malignancies, PTLD, and subsequent primary occurrences of NMSC after HT. These findings provided further explanation of the late survival benefit with long-term SRL use. Published by Elsevier Inc.