Sphingosine-1-phosphate (S1P) is a bioactive signalling molecule that mediates important cellular functions such as cell proliferation, cytoskeletal rearrangement, angiogenesis, mobilization of intracellular calcium, and immune cell trafficking. 2-Amino-2-[2-(4-octylphenyl) ethyl] propane-1,3-diol hydrochloride (also known as FTY720 or Fingolimod), a synthetic analog of sphingosine that has immunosuppressive properties, is the first oral drug approved by the U.S. FDA for treatment of multiple sclerosis. It is hypothesized that FTY720 is phosphorylated by sphingosine kinase 2 (SphK2) prior to binding to S1P receptor 1 (S1PR1) followed by internalization and degradation of the receptor. Here we examined the cellular uptake, partitioning, and diffusion of two fluorescent analogs of this drug (namely, Bodipy-FTY720) in cultured C3H10T1/2 cells, derived from mouse embryos. Multichannel imaging and co-localization with the endoplasmic reticulum (ER) tracker (Glibenclamide-Bodipy-FL) indicates that Bodipy-FTY720 resides in the ER of C3H10T1/2 cells, where it is expected to be phosphorylated by SphK2, and is excluded from the nucleus. This conclusion is supported by the rotational and translational diffusion measurements of this FTY720 analog in the ER membrane of the cells. Our results also indicate a heterogeneous environment of the cellular Bodipy-FTY720 as revealed by two-photon fluorescence lifetime imaging. These studies represent a step forward towards elucidating the effects of Bodipy moiety on the action mechanism of FTY720 at the single-cell level.