A simple and practical process was developed for the efficient separation of diastereomeric syn- and anti-2,4-pentanediol by selective acetalization of a diastereomeric mixture of the 2,4-pentanediols and selective hydrolysis of the corresponding acetals. The process relies upon the reaction rate differences of syn-2,4-pentanediol (syn-diol) and anti 2,4-pentanediol (anti-diol) in acetalization and of the corresponding acetals in hydrolysis: the syn-diol reacts faster to form a more stable acetal than the anti-diol, which in turn is more susceptible to hydrolysis by Brønsted acid. Acetalization of a 2,4-pentanediol diastereomeric mixture (syn/anti = 45:55) with acetophenone (0.95 equiv relative to syn-diol) leads to the formation of a syn-enriched acetal mixture with a syn/anti diastereomeric ratio (drs/a) of 6:1, leaving an anti-enriched diol mixture (drs/a = 1:7). Subsequent kinetic resolution via selective hydrolysis of the minor anti-acetal with a catalytic amount of 1.0 N HCl at ambient temperature affords the pure syn-acetal (drs/a > 99:1) in the organic phase and the anti-enriched 2,4-pentanediols (drs/a = 1:6) in the aqueous phase, which are conveniently separated by a phase cut. Hydrolysis of the syn-acetal is facile in alcohol solvents at elevated temperatures (60–80 °C), yielding the pure syn-diol. A second acetalization of the anti-enriched 2,4-pentanediols leads to the pure anti-2,4-pentanediol. This separation gives the syn-diol in 75–79% yield with drs/a > 99:1 and the anti-diol in 79–85% yield with dra/s > 98:2. Additionally, the acetophenone used for the acetalization can be recovered in 88–92% yield, and therefore, the overall process is high-yielding, atom-economical, and potentially recyclable.