Germany's Lisa Brennauer was edged into second as Vos came third to move three seconds clear of Christine Majerus of Luxembourg.
Majerus, who came fifth, had pipped Vos to victory on the opening leg of the five-stage race on Wednesday.
Lizzie Armistead was the highest-placed Briton, in 18th, on the 140km ride from Atherstone to Stratford-Upon-Avon.
The 27-year-old reigning world, Commonwealth and national road race champion took the Best British Rider jersey as she continues her preparations for the Olympic Games in Rio de Janeiro in August.
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1. Amy Pieters (Ned/Wiggle High5) 3hrs 36mins 55secs
2. Lisa Brennauer (Ger/Canyon-SRAM) same time
3. Marianne Vos (Ned/Raboliv) same time
4. Gracie Elvin (Aus/Orica-AIS) same time
5. Christine Majerus (Lux/Boel-Dolmans) same time
6. Emma Johansson (Swe/Wiggle High5) same time
7. Giorgia Bronzini (Ita/Wiggle High5) same time
8. Floortje Mackaij (Ned/Liv-Plantur) same time
9. Leah Kirchmann (Can/Liv-Plantur) same time
10. Anna van der Breggen (Ned/Raboliv) same time
Selected others
18. Lizzie Armitstead (GB/Boels-Dolmans) same time
19. Dani King (GB/Wiggle High5) same time
32. Laura Massey (GB/Drops) +1min 42secs
45. Jessie Walker (GB/British National) same time
46. Rebecca Durrell (GB/Drops) +4mins 57secs
48. Annasley Park (GB/British National) same time
53. Grace Garner (GB/British National) same time
1. Marianne Vos (Ned/Raboliv) 7hrs 1min 26secs
2. Christine Majerus (Lux/Boel-Dolmans) +3secs
3. Amy Pieters (Ned/Wiggle High5)) +7secs
4. Lisa Brennauer (Ger/Canyon-SRAM) +11secs
5. Giorgia Bronzini (Ita/Wiggle High5) +13secs
6. Leah Kirchmann (Can/Liv-Plantur) same time
7. Gracie Elvin (Aus/Orica-AIS) +14secs
8. Lizzie Armitstead (GB/Boels-Dolmans) +16secs
9. Floortje Mackaij (Ned/Liv-Plantur) +17secs
10. Emma Johansson (Swe/Wiggle High5) same time
Marianne Vos moved into the overall lead at the Women's Tour as her Dutch compatriot Amy Pieters won stage two.