Key: Win : Draw : Loss
| Date | Home | Away | Ref | ||
|---|---|---|---|---|---|
| 08/08/2009 | Southampton | 1 | 1 | Millwall | M Russell |
| 15/08/2009 | Huddersfield | 3 | 1 | Southampton | A Haines |
| 18/08/2009 | Swindon | 1 | 0 | Southampton | S Cook |
| 22/08/2009 | Southampton | 1 | 1 | Brentford | D Phillips |
| 29/08/2009 | Stockport | 1 | 1 | Southampton | C Boyeson |
| 05/09/2009 | Southampton | 0 | 0 | Colchester | G Scott |
| 12/09/2009 | Charlton | 1 | 1 | Southampton | K Wright |
| 19/09/2009 | Southampton | 2 | 0 | Yeovil | A Bates |
| 26/09/2009 | Carlisle | 1 | 1 | Southampton | G Salisbury |
| 29/09/2009 | Southampton | 2 | 3 | Bristol Rvs | D Deadman |
| 03/10/2009 | Southampton | 4 | 1 | Gillingham | S Attwell |
| 09/10/2009 | Southend | 1 | 3 | Southampton | G Hegley |
| 17/10/2009 | Oldham | 1 | 3 | Southampton | D Foster |
| 24/10/2009 | Southampton | 3 | 1 | Milton Keynes Dons | M Russell |
| 31/10/2009 | Leyton Orient | 2 | 2 | Southampton | J Linington |
| 15/11/2009 | Southampton | 1 | 3 | Brighton | K Friend |
| 21/11/2009 | Southampton | 2 | 2 | Norwich | A Penn |
| 24/11/2009 | Hartlepool | 1 | 3 | Southampton | N Swarbrick |
| 01/12/2009 | Southampton | 1 | 0 | Wycombe | D Whitestone |
| 05/12/2009 | Walsall | 1 | 3 | Southampton | C Pawson |
| 12/12/2009 | Southampton | 3 | 0 | Tranmere | D McDermid |
| 19/12/2009 | Leeds | 1 | 0 | Southampton | N Miller |
| 26/12/2009 | Southampton | 3 | 1 | Exeter | G Horwood |
| 28/12/2009 | Colchester | 2 | 1 | Southampton | D Deadman |
| 16/01/2010 | Millwall | 1 | 1 | Southampton | A Woolmer |
| 26/01/2010 | Brentford | 1 | 1 | Southampton | C Sarginson |
| 30/01/2010 | Southampton | 2 | 0 | Stockport | F Graham |
| 06/02/2010 | Exeter | 1 | 1 | Southampton | M Haywood |
| 20/02/2010 | Norwich | 0 | 2 | Southampton | R Booth |
| 23/02/2010 | Wycombe | 0 | 0 | Southampton | D Deadman |
| 27/02/2010 | Southampton | 5 | 1 | Walsall | G Scott |
| 02/03/2010 | Southampton | 5 | 0 | Huddersfield | P Miller |
| 06/03/2010 | Tranmere | 2 | 1 | Southampton | P Quinn |
| 13/03/2010 | Southampton | 1 | 0 | Leeds | P Walton |
| 16/03/2010 | Southampton | 0 | 1 | Swindon | K Hill |
| 20/03/2010 | Milton Keynes Dons | 0 | 3 | Southampton | C Webster |
| 23/03/2010 | Southampton | 3 | 2 | Hartlepool | A D'Urso |
| 01/04/2010 | Brighton | 2 | 2 | Southampton | S Tanner |
| 05/04/2010 | Southampton | 2 | 1 | Leyton Orient | G Ward |
| 10/04/2010 | Southampton | 1 | 0 | Charlton | G Hegley |
| 13/04/2010 | Bristol Rvs | 1 | 5 | Southampton | P Crossley |
| 17/04/2010 | Yeovil | 0 | 1 | Southampton | O Langford |
| 20/04/2010 | Southampton | 0 | 0 | Oldham | K Wright |
| 24/04/2010 | Southampton | 3 | 2 | Carlisle | S Hooper |
| 01/05/2010 | Gillingham | 2 | 1 | Southampton | M Russell |
| 08/05/2010 | Southampton | 3 | 1 | Southend | R Shoebridge |