Soil microbes drive biological functions that mediate chemical and physical processes necessary for plants to sustain growth. Laboratory soil respiration has been proposed as one universal soil health indicator representing these functions, potentially informing crop and soil management decisions. Research is needed to test the premise that soil respiration is helpful for profitable in-season nitrogen (N) rate management decisions in corn (Zea mays L.). The objective of this research was two-fold: (i) determine if the amount of N applied at the time of planting effected soil respiration, and (ii) evaluate the relationship of soil respiration to corn yield response to fertilizer N application. A total of 49 N response trials were conducted across eight states over three growing seasons (2014–2016). The 4-day Comprehensive Assessment of Soil Health (CASH) soil respiration method was used to quantify soil respiration. Averaged over all sites, N fertilization did not impact soil respiration, but at four sites soil respiration decreased as N fertilizer rate applied at-planting increased. Across all site-years, soil respiration was moderately related to the economical optimum N rate (EONR) (r2 = 0.21). However, when analyzed by year, soil respiration was more strongly related to EONR in 2016 (r2 = 0.50) and poorly related for the first two years (r2 < 0.20). These results illustrate the factors influencing the ability of laboratory soil respiration to estimate corn N response, including growing-season weather, and the potential of fusing soil respiration with other soil and weather measurements for improved N fertilizer recommendations.