Pedestrian, tram, and bus crossings, as well as northbound car travel from Mexico, will not be affected.
Tens of thousands of people pass through the border every day.
The $741m (£546m) expansion project is expected to cause traffic delays until it is completed in 2019.
The closure of southbound Interstate 5 and I-805 starts on Saturday at 03:00 (08:00GMT), and remains in effect until noon on Monday.
Vehicle travellers to Mexico have been advised to detour to the Otay Mesa Land crossing, about nine miles to the east, until the crossing reopens.
On any given day, San Ysidro processes an average of 50,000 northbound vehicles and 25,000 northbound pedestrians coming from Mexico.
The San Diego Association of Governments decided to undertake the project after estimating an 87% increase in vehicle traffic by the year 2030.
It aims to realign the I-5 motorway where it connects to Mexico's El Chaparral Port of Entry, and double the number of vehicle inspection lanes.
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