BACKGROUND This study aimed to determine the best size needle to use in inducing IVDD and to find the proper time point of disc degeneration suitable for further biologic treatment study. MATERIAL AND METHODS First, rat tail level 5/6, 7/8, and 9/10 discs were punctured by 18G, 21G, or 25G needles. Then, degeneration was assessed by radiography, MRI, and histological evaluation at 2, 4, and 6 weeks after puncture. Later, real-time reverse transcriptase (RT-PCR) was used to examine mRNA expressions of aggrecan, collagen type II, hypoxia-inducible factor-1a (HIF-1a), glucose transporter1 (GLUT-1), and vascular endothelial growth factor (VEGF). RESULTS Significant differences were identified in almost all parameters compared with the control group in the 18G and 21G group at almost all time points. To assess the effect of different needle sizes on DHI, we used magnetic resonance imaging (MRI), grade, and mRNA expression. We found significant differences between different groups, except for DHI between the 21G group and 25G group and MRI grade between the 18G and 21G group at the 2-week time point. In assessing the effect of different needle sizes on HE staining score and toluidine blue staining grade, statistical differences were observed at some time points. The effects of time on all parameters were significant at almost all time points in all groups. CONCLUSIONS The middle-size needle (21G) performed better in inducing disc degeneration. The 2-week time point may be better for use in further experimental studies.