The Argentina international spent five years with Atletico, scoring 74 goals in 175 games, and although some of those strikes came against the Galacticos it was never enough to secure victory.
Aguero hopes that drought will end in order to keep City's slim Champions League chances alive.
"It's always special to play against a club like Real Madrid," he said.
"Personally, my connection with Atletico Madrid gives it an additional kick - but there's no difference in how much I want to win this match.
"Never defeating Real when I was with Atletico was a real regret for me - Atletico haven't succeeded in beating them for many years - but I did get to score against them even if we never managed a win.
"Wednesday night will hopefully break that run for me."
Bottom of Group D with two points, five behind their opponents, City have to beat Real to avoid a second successive group stage elimination.
Even if they do prevail at the Etihad Stadium Roberto Mancini's side would then have to beat Borussia Dortmund and hope other results go their way.
"We should never give up. Where there's a will, there's a way, right?" Aguero told mcfc.co.uk.
"Of course, it's harder now because we're not just relying on ourselves but also on the other teams' results.
"I think it's even more significant now than if we had been in a better position in the table.
"It may sound strange but I firmly believe that. We must win this game.
"Whatever the final scoreline is - a good one, I hope - we'll show on the field that we don't give up, like we did in the game against Ajax and in every game we've played.
"We have a great team, I have no doubt about it and now it is time for us to show just how good we are.
"What we mustn't do is leave the pitch with any regrets and be content knowing that we have given our all.
"This club deserves nothing less and our supporters deserve as much, too."
Fellow Argentina striker Carlos Tevez is of a similar mindset, insisting they cannot afford to think their Champions League dreams are over for another year.
"We knew it would be tough," he told Champions - the official UEFA Champions League magazine.
"We only started playing in the competition last year so you need a bit of patience.
"You have to keep on growing and becoming more mature and, as the years pass, you will become stronger.
"We know it will be difficult but you just have to keep on fighting until the end."
After last year's fall-out with Mancini and extended stay in Argentina speculation about Tevez's future is never far from the headlines.
He appears much more settled this season, however, and Champions League football is helping with that.
"The truth is that I am happy when I am playing in games like the one against Madrid even though we couldn't get a good result," added the 2008 Champions League winner with Manchester United, referring to their 3-2 defeat at the Bernabeu in September.
"I am content, and you can see that on the pitch."
Mancini's team selection will be crucial on Wednesday and the Italian admits one of the key decisions will surround whether to play Yaya Toure in a deeper midfield position or not.
"If Yaya plays the way he did on Saturday (in a 5-0 win over Aston Villa), we can win a lot of games," said Mancini.
"He brought balance to the team, he didn't lose the ball, and was strong.
"The whole team played well but Yaya was important in the middle.
"He can make those runs from the holding position, but we can't forget that we have four offensive players in front and you need balance - without that balance it makes it difficult for the defenders."