<p>Myrosinase (β-thioglucoside glucohydrolase, EC 3.2.3.1) is a plant enzyme that hydrolyzes glucosinolates, which are introduced to soil by some plants into various compounds which can be toxic against plant pests. The positive effects of sulfur fertility on myrosinase activity in soils, particularly in soils cropped with <i>Brassica napus</i>, remains elusive. Here, we explore the key role of sulfur nutrition in the regulation of myrosinase activity in two contrasting soil types, and its further impact on the uptake of nutrients, chlorophyll, and growth of <i>Brassica napus</i>. <i>Brassica napus</i> plants were cultivated in both soils with various levels of sulfur. Sulfur concentrations were adjusted by diluting each soil with sand at ratios of 1:1, 1:3 and 1:9 and by addition of 0.2 or 0.5 g&#xa0;kg⁻¹ soil of composted farmyard manure to achieve different levels of sulfur. Plants grown in Hoytville clay loam soil with sulfur regime of 13.9–14.8 mg kg⁻¹ revealed higher uptake of nutrients. A sulfur supply of 8.9–13.9 mg kg⁻¹ in Hoytville soil considerably increased chlorophyll <i>a</i> (1.13–1.15 mg g⁻¹) and chlorophyll<i> b</i> (0.66–0.69 mg g⁻¹) concentrations. The sulfur status of 13.9–14.8 mg kg⁻¹ in Hoytville soil and 9.8–12.0 mg kg⁻¹ in Wooster silt loam soil showed timely completion of the phenological growth stages. Plants supplemented with sulfur of 14.8 mg kg⁻¹ resulted in maximum shoot dry biomass (7.98 g) and a higher level of myrosinase activity (277 µg glucose g⁻¹ soil 4 h⁻¹) in Hoytville than Wooster soils. Hence, this study reveals myrosinase enzyme activity in soil is increased due to enhanced growth of <i>Brassica napus</i> brought about by the improved sulfur soil fertility.</p>