This study focused on preparation and evaluation of a thermosensitive and mucoadhesive in situ gelling ophthalmic system of azithromycin (ATM). Poloxamer 407 (P407) and poloxamer 188 (P188) were used as gelling agents. Addition of Carbopol 974P (CP 974P) to the gelling systems could increase the solubility of ATM by salt effect and enhance the mucoadhesive property of the systems. Gelation temperature of these systems ranged from 31.21-36.31 degrees C depending on the ratio of P407 and P188. Mucoadhesion force of the system composed of P407/P188/CP 974P (21/5/0.3%, w/v) was 2.3-fold that without carbopol 974P. Viscosity of the formulation was in a suitable range at 25 degrees C and pseudoplastic behavior was observed at 35 degrees C. The formulation exhibited a 24-h sustained release of ATM. In vivo resident experiments showed AUC(0-12) of ATM in rabbit tears increased by 1.78-fold for in situ gel compared with eye drop. At 12 h, tear concentrations exceeded minimum inhibitory concentration (MIC) breakpoint for the most common causative pathogens of bacterial conjunctivitis by 2.8-fold. Results in vitro and in vivo indicated that this droppable gel performed better than ATM eye drop did.