To investigate whether the non-structural protein 2B of rhinovirus 16 induces endoplasmic reticulum stress (ERS) and apoptosis, BHK-21 cells were transfected with a plasmid p2B-GFP expressing rhinovirus 16 2B protein. Biomarkers of ERS and apoptosis were detected respectively. The results showed that 2B protein was localized to the ER apparatus. 2B protein increased the expressions of ERS marker proteins Grp78 and CHOP and the transcriptional activity of activating transcription factor 6 (ATF6). We also found 2B protein induced nuclear condensation associated with apoptosis and degradation of marker protein PARP in BHK-21 cells. In conclusion, the rhinovirus 16 2B protein induces both ERS and apoptosis.