The aim of this work is to explore effects of carrageenan on sustained-release properties of poloxamer 407-based vaginal in situ gel. After formulation of composite gel systems composed of carrageenan and poloxamer 407, in vitro release profiles and in vivo local drug residence after vaginal administration in mice was investigated using acyclovir as the model drug. Rheological experiment was conducted to investigate effects of carrageenan on temperature-dependent viscoelasticity of poloxamer 407-based gels. It appeared that carrageenan and poloxamer 407 could form composite gel systems with good thermosensitity similar to gels containing only poloxamer 407. In the in vitro release experiment, carrageenan significantly decreased the release rate of acyclovir, retarded the dissolution of poloxamer 407 and slowed the gel erosion (weight loss) in a concentration-dependent manner. In vivo local drug residence experiment indicated that carrageenan significantly prolonged local residence of acyclovir and further showed a synergistic bioadhesive effect with acrylic acid polymers (Carbopol). In conclusion, carrageenan was able to improve the sustained-release properties of poloxamer 407-based in situ gel, indicating that the combination of carrageenan and poloxamer 407 may find use in the development of vaginal in situ gel drug delivery systems with prolonged local residence and thus better clinical outcome.