Nighttime crashes are over-represented on the highway system in the United States. Roadway lighting, providing additional visibility by supplementing vehicle headlights, has been identified as an effective countermeasure to improve nighttime safety. However, the effects of street lighting illuminance parameters in reducing nighttime crashes on roadway segments are not well-documented. This study aimed to investigate the effects of street lighting illuminance, rather than the presence of street lighting, on nighttime crash occurrence on roadway segments. Illuminance data were collected from 403 roadway segments in the Tampa Bay area in Florida from 2012–2014 using the Advanced Lighting Measurement System matched with four years of crash data (2011–2014). Zero-inflated negative binomial models were developed based on the collected data for addressing the issues of over-dispersion and excessive zero observations in the sample. The major conclusions include the following: (1) mean horizontal illuminance significantly and near-logarithmically reduces nighttime crash frequency and night-to-day crash ratio; (2) good illuminance uniformity (max/min < 6) can significantly reduce expected nighttime crash occurrence by 24.6% (based on expected nighttime frequency) or 2.3% (based on night-to-day ratio), compared to poor uniformity (max/min ≥ 6); (3) compared to the night-to-day crash ratio, the expected nighttime crash frequency-based analysis tends to overestimate the safety performance of average illuminance; and (4) considering that the night-to-day crash ratio can hedge the influence from the confounding or interacting variables that are related to illuminance, the CMF based on the night-to-day crash ratio is preferred.