George Bowerman levelled things up on 89 minutes before Matt Benham's free-kick found the far corner deep in stoppage-time to inflict a 2-1 defeat on a shell-shocked USP.
With just over a minute of normal time to go, it looked as though Tom Grice's men were on track to claim a fourth successive league triumph.
Leading scorer Joe Johnson, now on nine goals for the season, had fired the hosts ahead in the first-half and seemingly on the path to an eighth Wessex Premier win in 20 matches.
However, Alton's remarkable late show left US Portsmouth on their knees and completely stunned.
Dan Sibley and Jack Chandler had fired a couple of early warnings to Alton before the hosts took the lead.
Johnson timed his run to perfection, beating the offside trap, then firing into the far corner after 23 minutes.
USP's leading scorer was then guilty of missing a good chance while Lamin Jatta's cross just evaded Cameron Quirke as US Portsmouth looked to get a crucial second goal.
But failure to double their advantage would prove costly, with Bowerman levelling things up on 89 minutes.
US Portsmouth would probably have been disappointed just to come away with a point, yet disaster struck five minutes into stoppage-time as Benham's curling free-kick found the net to leave Grice and his troops stunned.