Background: This review aims to describe the activities of nurse practitioners (NPs) and physician assistants (PAs) working in ambulance care, and the effect of these activities on patient outcomes, process of care, provider outcomes, and costs. Methods: PubMed, MEDLINE (EBSCO), EMBASE (OVID), Web of Science, the Cochrane Library (Cochrane Database of Systematic Review), CINAHL Plus, and the reference lists of the included articles were systematically searched in November 2019. All types of peer-reviewed designs on the three topics were included. Pairs of independent reviewers performed the selection process, the quality assessment, and the data extraction. Results: Four studies of moderate to poor quality were included. Activities in medical, communication and collaboration skills were found. The effects of these activities were found in process of care and resource use outcomes, focusing on non-conveyance rates, referral and consultation, on-scene time, or follow-up contact Conclusions: This review shows that there is limited evidence on activities of NPs and PAs in ambulance care. Results show that NPs and PAs in ambulance care perform activities that can be categorized into the Canadian Medical Education Directives for Specialists (CanMED) roles of Medical Expert, Communicator, and Collaborator. The effects of NPs and PAs are minimally reported in relation to process of care and resource use, focusing on non-conveyance rates, referral and consultation, on-scene time, or follow-up contact. No evidence on patient outcomes of the substitution of NPs and PAs in ambulance care exists. PROSPERO registration: CRD42017067505 (07/07/2017)