Lymphatic endothelial cells (LECs) play a critical role in the formation and maintenance of the lymphatic vasculature, which is essential for the immune system, fluid balance, and tissue repair. However, LECs are often difficult to study in vivo and in vitro models that accurately mimic their behaviors and phenotypes are limited. In particular, LECs have been shown to lose their lymphatic markers over time while being cultured in vitro, which reflect their plasticity and heterogeneity in vivo. Since LECs uniquely express lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), we hypothesized that surface coating with hyaluronic acid (HA) can preserve LEC phenotypes and functionalities. Dopamine conjugated hyaluronic acid (HA–DP) was synthesized with 42% degree of substitution to enable surface modification and conjugation onto standard tissue culture plates. Compared to fibronectin coating and tissue culture plate controls, surface coating with HA–DP was able to preserve lymphatic markers, such as prospero homeobox protein 1 (Prox1), podoplanin (PDPN), and LYVE-1 over several passages in vitro. LECs cultured on HA–DP expressed lower levels of focal adhesion kinase (FAK) and YAP/TAZ, which may be responsible for the maintenance of the lymphatic characteristics. Collectively, the HA–DP coating may provide a novel method for culturing human LECs in vitro toward more representative studies in basic lymphatic biology and lymphatic regeneration.