Ultrasonication (US) is an emerging technology being applied to dairy streams. The objective of this study was to determine the effect of US on reconstituted deproteinized whey (DPW) before crystallization and isolation of lactose. Commercial DPW was reconstituted to 65% TS (wt/wt) at 80°C. Solutions were then seeded (0.027 g/100 g solution) with lactose crystals, or treated with US (10 min, 22.5 Hz) and then seeded. Each sample was then stirred for one min and transferred into a 1 L glass crystallizer (CST). The CST was immersed into a temperature controlled bath and cooled from 80 to 18°C (rate = −0.0479°C/min). Constant agitation (100 rpm) was applied to the CST by an overhead stirrer and agitator. After crystallization, approx. 225 g of solution was transferred to a centrifuge bottle. The solution was centrifuged at 10,000 × g for 30 min at 5°C. The supernatant was decanted, weighed, and a mass of deionized water (<4.0°C) equivalent to the supernatant was added to resuspend the crystals before centrifugation. The resuspended crystals were centrifuged, decanted, and resuspended 3 more times using the previous procedure (4 washes total). Recovered lactose crystals (>75 crystals) were measured microscopically (10×) to determine mean crystal size. Total solids of commingled supernatants, and of isolated crystals were measured to assess yield. Each treatment was repeated in triplicate. Mean crystal size, and lactose yield data were analyzed for statistical significance with replicate and treatment as categorical explanatory variables. Mean crystal size was larger (P < 0.01) for the US treatment, 85.0 µm, than for the control treatment, 74.4 µm. Lactose yields, 78.7 and 78.9%, for control and US, respectively, were not different (P > 0.05). Theoretical yield for lactose at the conditions used is 88%. Yields may be overestimated due to non-lactose impurities. Ultrasonication with seeding of reconstituted DPW before crystallization increased lactose crystal size, but did not increase lactose yield.