Cold stress seriously inhibits plant growth and development, geographical distribution, and yield stability of plants. Cold acclimation (CA) is an important strategy for modulating cold stress, but the mechanism by which CA induces plant resistance to cold stress is still not clear. The purpose of this study was to investigate the effect of CA treatment on the cold resistance of citrus seedlings under cold stress treatment, and to use seedlings without CA treatment as the control (NA). The results revealed that CA treatment increased the content of photosynthetic pigments under cold stress, whereas cold stress greatly reduced the value of gas exchange parameters. CA treatment also promoted the activity of Rubisco and FBPase, as well as led to an upregulation of the transcription levels of photosynthetic related genes (
rbcL
and
rbcS
),compared to the NA group without cold stress. In addition, cold stress profoundly reduced photochemical chemistry of photosystem II (PSII), especially the maximum quantum efficiency (F
v
/F
m
) in PSII. Conversely, CA treatment improved the chlorophyll
a
fluorescence parameters, thereby improving electron transfer efficiency. Moreover, under cold stress, CA treatment alleviated oxidative stress damage to cell membranes by inhibiting the concentration of H
2
O
2
and MDA, enhancing the activities of superoxide dismutase (SOD), catalase (CAT), ascorbic acid peroxidase (APX) and glutathione reductase (GR), accompanied by an increase in the expression level of antioxidant enzyme genes (
CuZnSOD1
,
CAT1, APX
and
GR
). Additionally, CA also increased the contents of abscisic acid (ABA) and salicylic acid (SA) in plants under cold stress. Overall, we concluded that CA treatment suppressed the negative effects of cold stress by enhancing photosynthetic performance, antioxidant enzymes functions and plant hormones contents.