Purpose：In the present study, we aimed to compare and analyze the clinical features, diagnosis, treatment and prognosis of primary pyogenic spondylitis caused by Gram-positive and Gram-negative bacteria.Methods: A retrospective analysis consisting of 76 cases of primary pyogenic spondylitis with complete clinical information was carried out from January 2013 to January 2020 in our hospital. The patients were divided into two groups according to Gram staining: Gram-negative group (n=33) and Gram-positive group (n=43). The clinical characteristics, diagnosis, treatment and prognosis of the two groups were compared and analyzed.Results: Staphylococcus aureus accounted for the highest proportion of the Gram-positive group, while Escherichia coli accounted for the highest proportion of the Gram-negative group. Before treatment, there were no significant differences in terms of age, gender, affected segment, spinal abscess, diabetes mellitus, course of disease, admission erythrocyte sedimentation rate (ESR), admission C-reactive protein (CRP), and admission white blood cell (WBC) count between the two groups (P>0.05). After treatment, there were no statistically significant differences in discharge ESR, discharge CRP, ESR decline rate, CRP decline rate, surgery, recurrence, follow-up time, hospital stay, and body temperature ≥38℃ between the two groups (P>0.05). The body temperature of the Gram-negative group was higher compared with the Gram-positive group, and the number of patients with urinary tract infection in the Gram-negative group was significantly greater compared with the Gram-positive group (P<0.05). Antibiotic treatment time <6 weeks was an independent risk factor for recurrent infection.Conclusions: The body temperature of the Gram-negative group was higher compared with the Gram-positive group, and there were significantly more cases with urinary tract infection in the Gram-negative group compared with the Gram-positive group (P<0.05). Antibiotic treatment time <6 weeks was an independent risk factor for recurrent infection.