Recent studies have identified phagocytic B cells in a variety of species, yet little is understood about their function and how it is influenced by natural environmental variation, such as temperature. Phagocytic B-cells are present in red-eared slider turtles, Trachemys scripta, and the wide range of temperatures experienced by these ectotherms may have an effect on immunity, including B cell antibody secretion and phagocytosis. We examined the impact of environmental temperature on B cell function in vitro using phagocytic and ELISpot assays conducted at biologically relevant temperatures. We found a significant effect of temperature on antibody secretion, with maximal antibody secretion occurring at intermediate temperatures (estimated maximum of 28.8 °C). There was no effect of temperature on phagocytosis. We also noted a difference in the efficiency of phagocytosis in this assay between B cells and non-B cells. Interestingly, in our in vitro assay, phagocytic B cells engulfed more foreign fluorescent beads per cell than phagocytes lacking surface immunoglobulin. This work sheds light on our understanding of phagocytic B cells and the importance of environmental temperature on the behavior of reptilian immune cells, which may have relevance for organismal fitness.