Floral nectar proteins (nectarins) are mainly enzymes and play important roles in inhibiting microbial growth in nectar and tailoring nectar chemistry before or after secretory. Nectar proteomes are usually small, but only very few plant species have had their nectar proteomes thoroughly investigated. Nectarins from Nicotiana tabacum (NT) were separated using two-dimensional gel electrophoresis, and then analyzed using mass spectrometry. Glycoproteins were isolated from raw NT nectar, separated by SDS-PAGE, and identified by mass spectrometry. All eight identified nectarins and four invertase genes’ expression were analysed by qPCR. Sugars composition, total sugar concentration, protein content, polyphenol content and hydrogen peroxide content were compared at different time intervals in extracted nectar and nectar in situ after secretion. Totally, eight nectarins were detected in NT nectar in which only two are glycoproteins, beta-xylosidase and a protein with unknown function. All of the eight nectarin genes expression was not nectary-specific and not synchronous along with the nectary development. After secretion, NT nectar in flower tube changed from sucrose–rich to hexose-rich type even though no free invertase or its activity was detected in NT nectar. No sugar composition changes observed in extracted nectar after incubating at 30 ℃ up to 48 hours in plastic tubes. Our results indicate that nectar post-secretory changes could be a complex process and tissue closely contact with nectar might function in it.