STUDY DESIGN: Prospective cohort study. PURPOSE: To compare intraoperative parameters, radiation exposure, and pedicle screw perforation rate in navigation-guided versus non-navigated fluoroscopy-assisted minimal invasive transforaminal lumbar interbody fusion (MIS TLIF). OVERVIEW OF LITERATURE: The poor reliability of fluoroscopy-guided instrumentation and growing concerns about radiation exposure have led to the development of navigation-guided instrumentation techniques in MIS TLIF. The literature evaluating the efficacy of navigation-guided MIS TLIF is scant. METHODS: Eighty-seven patients underwent navigation- or fluoroscopy-guided MIS TLIF for symptomatic lumbar/lumbosacral spondylolisthesis. Demographics, intraoperative parameters (surgical time, blood loss), and radiation exposure (sec/mGy/Gy.cm² noted from C-arm for comparison only) were recorded. Computed tomography was performed in patients in the navigation and non-navigation groups at postoperative 12 months and reviewed by an independent observer to assess the accuracy of screw placement, perforation incidence, location, grade (Mirza), and critical versus non-critical neurological implications. RESULTS: Twenty-seven patients (male/female, 11/16; L4–L5/L5–S1, 9/18) were operated with navigation-guided MIS TLIF, whereas 60 (male/female, 25/35; L4–L5/L5–S1, 26/34) with conventional fluoroscopy-guided MIS TILF. The use of navigation resulted in reduced fluoroscopy usage (dose area product, 0.47 Gy.cm² versus 2.93 Gy.cm²), radiation exposure (1.68 mGy versus 10.97 mGy), and fluoroscopy time (46.5 seconds versus 119.08 seconds), with p-values of < 0.001. Furthermore, 96.29% (104/108) of pedicle screws in the navigation group were accurately placed (grade 0) (4 breaches, all grade I) compared with 91.67% (220/240) in the non-navigation group (20 breaches, 16 grade I+4 grade II; p=0.114). None of the breaches resulted in a corresponding neurological deficit or required revision. CONCLUSIONS: Navigation guidance in MIS TLIF reduced radiation exposure, but the perforation status was not statistically different than that for the fluoroscopy-based technique. Thus, navigation in nondeformity cases is useful for significantly reducing the radiation exposure, but its ability to reduce pedicle screw perforation in nondeformity cases remains to be proven.