It serves local traffic in nearby cities including La Crosse, Tomah, Wisconsin Dells, Portage, Columbus, Watertown, Oconomowoc and Waukesha.

WIS 16 passes the northern edge of the University of Wisconsin–La Crosse campus, then turns north at Losey Boulevard.

The route then follows I-90 very closely as a frontage road to Tomah where it junctions with WIS 131 and joins US 12.

The routes slowly trek eastward and pass through Lyndon Station where Rocky Arbor State Park is located.

The highways turn southeast again and crossing the Interstates and into Sauk County and Wisconsin Dells.

The highway then passes through Portage, crossing WIS 33 and joining US 51 south for five miles (8.0 km).

WIS 16 passes around Ixonia, becomes an expressway, and enters Waukesha County along a bypass of Oconomowoc.

The current eastern terminus of WIS 16 was the site of the dedication ceremonies for the first completed segment of Interstate 94 in Wisconsin.

The first major interchange on the new Interstate was originally where US 16 turned north towards Pewaukee and WIS 30 continued west with I-94.

[1] The alignment of the eastern section of Highway 16 changed in late 2006 when the rest of the Oconomowoc bypass was opened to traffic.

Eventually, the bypass will result in the removal of the state highway designation from Wisconsin Avenue through downtown Oconomowoc.

[14] As of November 2008, Oconomowoc officials and WisDOT had not yet set a firm date for the jurisdictional transfer, though it was likely that it will be done by the summer of 2009.