Objective To develop a scan sequence to reduce respiratory motion and heartbeat artifacts in 256-slice spiral CT in living rat chest CT scans. Methods Using a Philips iCT, chest CT imaging was performed on 10 rats.For each rat, both A and B scanning methods were used: A mode for a conventional chest CT scan sequence, non-ECG gating spiral scanning mode, and B mode for non-ECG gating axis scanning mode. Both scanning method were performed under 120 kV and the scan length was 8 cm. Reconstruction of transverse and coronal lung windows and soft-tissue windows was performed on the obtained CT data. The images were observed and measured. Differences in mean CT values, image noise, signal-to-noise ratio (SNR), and subjective image quality scores of the two groups of images were analyzed. In addition, the consistency of the subjective image quality scores was tested among different observers. Results A total of 10 sets of rat chest CT data were analyzed. The DLP values of the scanning sequences of A and B were 144. 7 mGy/ cm and 72. 2 mGy/ cm. There was no significant difference between the CT values of lung tissue and of liver tissue between the two groups (( t = - 0. 205 and 0. 545; P > 0. 05). There was no statistically significant difference in noise between the two groups (( t = - 0. 865, P > 0. 05). There was also no statistically significant difference in lung tissue SNR between the two groups (( t =0. 903, P > 0. 05). However, liver SNR was significantly higher in group B than in group A (( t = - 2. 885, P <0. 05). The correlation coefficient between the scores of two physicians was 0. 763, and the consistency of the diagnostic result was good. The subjective quality scores in groups A and B were 2. 5 ± 0. 53 points and 4. 3 ± 0. 67 points, which were significantly different (χ² = 14. 76, P < 0. 05). Conclusions The non-ECG gating axial scan mode can obtain approximately the same CT value, noise, and SNR as conventional chest CT scan (non-ECG gating spiral scan mode), but the use of non-ECG gating axial scan mode can effectively reduce respiratory and heartbeat artifacts in living rat chest CT scans, reduce radiation dose, improve image quality, and improve observers’ diagnostic confidence.