Chikungunya virus (CHIKV) is an arbovirus from the Togaviridae family and alphavirus genus. CHIKV is transmitted by the bites of infectious Aedes mosquitoes, and its genome is composed of a positive-sense single-stranded RNA. In order to spread, CHIKV needs to successfully infect a variety of cell types in both the insect vector and the vertebrate host. As an enveloped virus, the lipids present in the viral envelope are essential for mediating many steps of the viral replication cycle including entry and release. Phospholipids, such as phosphatidylserine (PS), are abundant at the plasma membrane and can be incorporated into viral envelopes at the site of budding. Phosphatidylserine receptors (PSR), including the T-cell immunoglobulin and mucin domain (TIM) proteins, interact with enveloped viruses playing a role in multiple stages of their replication cycle. PS in enveloped viral particles can mediate the attachment to host cells by binding to PSR on the surface of the cells during entry and release. Despite the identification of multiple factors mediating CHIKV entry, no essential receptor has been determined. In an effort to characterize the role of PS in the viral replication cycle of CHIKV we aimed to 1) evaluate CHIKV’s use of phospholipids during viral entry and their influence on viral infectivity using a panel of mammalian and mosquito cell lines, and 2) determine the ability of PS receptors to impede the efficient release of CHIKV particles and identify viral counteracting mechanisms. We used recombinant viral particles tagged with a luminescence reporter gene, nano-luciferase, in a separate open-reading frame or directly attached to the envelope protein E2 to facilitate quantification. We found that CHIKV’s use of attachment factors for mediating entry was highly cell line dependent. The production of viral particles high in envelope PS increases CHIKV’s infectivity in Vero cells but not in cells lacking PSR. In contrast, the presence of PS receptors on the surface of the infected cells significantly reduced CHIKV’s release efficiency. Altogether, this study highlights the importance of PS and PSR as modulators of CHIKV’s entry and release providing insight into the molecular mechanisms enabling optimal infection.