A known sweet pepper cDNA clone, CaHSP26 encoding the chloroplast-localized small heat shock protein (CPsHSP), was isolated and introduced into tobacco plants. It has been reported that CaHSP26 is a member of the CPsHSP gene family related to extreme temperature tolerance in plants. In the present work, the transcripts were detected in the transgenic tobacco lines. The actual quantum yield of photosynthesis (ΦPSII), non-photochemical quenching, and stomatal conductance (gs) in the transgenic lines overexpressing CaHSP26 were higher than those in the wild-type plants under a range of photosynthetic photon flux density during chilling stress. Electron microscopic analysis showed that the transgenic line (L1) had larger size of stomata to lessen stomatal limitation. The activities of ascorbate peroxidase (APX), peroxidase (POD) and catalase (CAT) were also higher in the transgenic lines than those in wild-type plants. Additionally, a significant increase in cis-unsaturated fatty acid contents was observed in transgenic lines due to lower temperatures. These results suggested that CaHSP26 protein plays an important role in protection of PSII by maintaining the antioxidative enzyme activities to avoid or mitigate photooxidation and increasing the fluidity of the thylakoid membrane during chilling stress under low irradiance. Key message CaHSP26 protein protects PSII by maintaining the antioxidative enzyme activities to avoid or mitigate photooxidation and increases the fluidity of the thylakoid membrane during chilling stress under low irradiance.