The Dane, who won the title in Eastbourne last week and was seeded second in London, lost 6-4 1-6 7-5 to the Russian on Court One.
Wozniacki’s exit means half of the top 10 seeds have already been dumped out in the opening two rounds. She follows Sloane Stephens, Elina Svitolina, Caroline Garcia and Petra Kvitova on her way home.
Some great hitting allowed unseeded Makarova to steam into a 5-1 lead in the first set, which she eventually took 6-4.
Wozniacki is renowned for her fighting spirit and looked to have gained the upper hand after cruising to the second set to level up.
Makarova regrouped, though, and surged to a 5-1 lead in the decider before having to fend off another fightback.
She served for the match, then had four match points at 5-4 before eventually getting the job done at the sixth attempt.
Makarova said: “Caroline is always a very tough opponent and we have played so many times.
“It’s only the second time I have beaten her. It is tough to face her but I am so happy.”