This study evaluated in vitro and in vivo a colloidal nanosystem with the potential to deliver drugs to the ocular surface. This nanosystem, liposome-chitosan nanoparticle complexes (LCS-NP), was created as a complex between liposomes and chitosan nanoparticles (CS-NP). The conjunctival epithelial cell line IOBA-NHC was exposed to several concentrations of three different LCS-NP complex to determine the cytotoxicity. The uptake of LCS-NP by the IOBA-NHC conjunctival cell line and by primary cultured conjunctival epithelial cells was examined by confocal microscopy. Eyeball and lid tissues from LCS-NP-treated rabbits were evaluated for the in vivo uptake and acute tolerance of the nanosystems. The in vitro toxicity of LCS-NP in the IOBA-NHC cells was very low. LCS-NPs were identified inside IOBA-NHC cells after 15 min and inside primary cultures of conjunctival epithelial cells after 30 min. Distribution within the cells had different patterns depending on the LCS-NP formulation. Fluorescence microscopy of the conjunctiva revealed strong cellular uptake of LCS-NP in vivo and less intensive uptake by the corneal epithelium. No alteration was macroscopically observed in vivo after ocular surface exposure to LCS-NP. Taken together, these data demonstrate that LCS-NPs are potentially useful as drug carriers for the ocular surface.