Implied in the poem is the author's recognition that the path 'less travelled by' was the best one to have taken. Instead of following the crowd, at some point in life he chose to strike out on his own. This path was less sure, more difficult, but at least there he left a greater imprint of his passing than he would have done along the more-travelled way.
Note that a path in the woods is a universal symbol. People from all cultures understand it to mean an individual's way through life. In the same manner, a fork along that way means a point where a choice between two 'destinations' must be made. This could be a career orientation, opting for ceibacy instead of marriage, embracing a religious belief, or whatever. The only thing the reader does know is that this choice was countercurrent to the 'normal' or usual trend, and that the author in the end was glad to have made such a decision.
Robert Frost published "The Road Not Taken" in 1916 in his collection "Mountain Interval." The last line can be interpreted both literally and ironically.
1. Literal meaning: "And that has made all the difference" emphatically states that the 'choice' one has made is important and not the opportunites that one has sacrificed. Thus the whole poem can be understood as a glorification of individualism. The traveller is a non-conformist who claims that he has done the right thing by "taking the road less traveled by." Thus, the poem inspires and encourages its readers to be unconventional.
2. Irony: The traveler regrets that he has made the wrong choice by "taking the road less traveled by." In the 2nd and 3rd stanza the poet admits that both the paths are equally worn and are equally leafy covered: "just as fair" and "equally lay in leaves." Hence, actually the choice has made no difference at all even though the speaker might confidently state otherwise.
The poem is deliberately ambiguous because when a person arrives at a fork in a road he becomes confused at the ambiguous choices in front of him. We come across many forks in the road of our journey called life and we are faced with tough choices which either challenge us to do something different or confuse and demoralise us.