The two Belgians both went for the same header with Vertonghen heading his team-mate.
The 32-year-old received lengthy treatment before changing his shirt.
Vertonghen returned to the pitch for Spurs but just moments later he signalled he could no longer continue and appeared to nearly collapse on the sidelines in distressing scenes.
Moussa Sissoko was the man to replace the Belgian as Mauricio Pochettino reorganised his side.
Ajax started the better of the two sides taking an early lead through Donny van de Beek.
Tottenham seemed to kick into action after falling behind but the sickening collision which saw the end of Vertonghen’s game left them reeling again.
BBC Sport pundit Dion Dublin was sad for Vertonghen and the way his night came to an end.
He said: “It’s such a pity. You can see Jan Vertonghen wanted to get back on the pitch.
“They patched him up and he put his clean shirt on but he does not look good.”
Vertonghen was left spitting blood but was determined to carry on in the first leg of Spurs’ Champions League semi-final.
Fans expressed their concern over the Belgian being allowed to return to the field at all.
One tweeted: “Genuinely hope Vertonghen is ok. That looked a really nasty head injury. Cannot believe the medical team allowed him back on! You could see clearly that he was all over the place. Shocking.”
Another said: “I say it alot but theres so much football can learn from rugby. Vertonghen should have been taken for a HIA (head injury assessment) with a replacement brought on for the duration. If passed fit, he comes back on, if not, the sub is permanent.”