Poloxamer 407 (P-407) composed of a poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG) unit has two distinct microenvironments: the interior core formed by the PPG unit and the exterior shell formed by the PEG unit. In this work, we have used two fluorescent molecules coumarin-153 and 8-anilino-1-naphthalene sulfonic acid (ANS) of contrasting natures to characterize and probe the water dynamics in the core and corona regions of the copolymer by means of spectroscopic techniques, namely, absorption, fluorescence, and time-resolved fluorescence emission spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Changes in the surface morphologies were characterized by using microscopic techniques. Further, two classes of osmolytes kosmotropic (betaine and sarcosine) and chaotropic (urea) known to perturb the water structure were added to aqueous solutions of P-407. Our studies reveal that the addition of kosmotropes decreases the critical micelle temperature (CMT) of the copolymer, whereas the chaotropic osmolyte increases the CMT. Steady-state studies reveal that the addition of the osmolytes to the copolymer increases the polarity of the micelle formed and hence results in the red shift in the ANS absorbance maximum. FTIR spectroscopy reveals that kosmotropes interact with the PEG moiety of the copolymer, whereas the chaotrope interacts with both the PEG and PPG moieties of the copolymer. Solvent relaxation studies produced less changes upon the addition of the kosmotropes, whereas a greater change in the relaxation time was observed in the presence of the chaotrope.