The City of Alachua continued to grow, albeit slowly. The economy was impacted by the construction of US Highway 441 in the 1940s, which moved traffic off the Dixie Highway and therefore bypassed Main Street businesses. The construction of Interstate-75 in the 1960s west of the City made access to Gainesville easier and faster, further shifting commerce away from local businesses.

Still, the community slowly grew. In 1950 the population was 1,116 and by 1960 it had grown to 1,974. In 1970, the U.S. Census listed it at 2,252.

Much of the downtown area had become blighted by the late 1970s and early 1980s, which is when community leaders made a concerted effort to revitalize the city. The Community Redevelopment Agency was founded in 1982. Main Street was redesigned - what had been a straight thoroughfare was changed to a more winding road, which encouraged traffic to slow down and visit local businesses. Commerce along Main Street began to pick up, and by 1995, Alachua was named Florida's Outstanding Rural Community of the Year.

That same year, the University of Florida's Sid Martin Biotech Incubator opened in Progress Park along the eastern US 441 corridor of Alachua, and what has now become one of Florida's largest bio and life science business hubs was born. Multiple businesses have successfully graduated from Sid Martin and established themselves in the Progress District. Combined with the several distribution centers that began establishing themselves on the western edge of the city at the same time, Alachua is now a leading job producer within North Central Florida.

Population has skyrocketed commensurately, more than doubling from 4,529 at the time of the 1990 Census to 10,574 in 2020, making Alachua the second-largest city in the county and third-largest in North Central Florida. With the continued development of the Progress District, the formation in 2019 of San Felasco Tech City's mixed-use business and residential ecosystem, and the growth of Santa Fe College's Perry Center for Emerging Technologies, Alachua is poised to further grow into a leading city within the region and state in the 21st Century.