The first stanza establishes the poem’s scenario and the attitude of the speaker towards the mouse. As the poem's subtitle indicates, the speaker has just destroyed the mouse’s nest with his plough. The speaker addresses the mouse in humorous, good-natured terms, as a "Wee" ("little") "sleeket, cowran, tim’rous beastie." The use of the affectionate "wee" as well as the diminutive terms “beastie” and “breastie” suggest that the speaker might be laughing a bit at the mouse. So does his exclamation "O" in line 2, as if he is treating the tiny animal with mock seriousness. But the reader quickly sees that the laughter is kind rather than malicious.
The speaker exclaims at the mouse's "panic" but quickly reassures her that she need not "start awa sae hasty," or run away so quickly, for he has no wish to "chase [her] / Wi' murd'ring pattle!" The speaker destroyed the mouse's nest only by accident, not on purpose. He has no wish to harm her. He is laughing at her because her panic is unnecessary, not because he finds any cruel pleasure in the animal's fright or his power over her.
The fact that the speaker is addressing his words to the mouse establish the poem's anthropomorphism. The speaker speaks to the mouse as though she can understand him. He also imagines the thoughts and feelings she is having, thoughts and feelings similar to those a human being would have. These actions imply that the speaker sees the mouse, in some sense, as being similar to a person or even on par with one in terms of how he ought to treat her. This implication will be further developed as one of the most important themes of the poem.
The colloquial language, vowel sounds, and rhymes add to the tone of gentle good humor in this first stanza. The lines are dominated by Scottish dialect, including archaic speech forms (thy, thou), variants on standard English words ("awa" for "away," "sae" for "so"), and Scottish words ("bickerin brattle"). The dialect shows the speaker addressing the mouse in casual, familiar terms.
The opening also lines repeat the long /ee/ sound (assonance) in "Wee," "sleeket," "beastie," "breastie," "need," "hasty," and "thee." Rhyming words and sounds can (though do not necessarily) add a comic sense to verse, especially when the rhymes are very frequent. Similarly, feminine line endings (that is, final, unstressed beats) can add a sense of humor or lightness, by avoiding the sense of seriousness and gravity that comes with ending the line on a stressed syllable. Lines 1-4 scan like this:
Wee, slee- | ket, cow- | ran, tim’- | rous beastie,
O, what | a pan- | ic’s in | thy breastie!
Thou need | na start | awa | sae hasty,
Wi’ bick- | erin brattle!
Altogether, the speaker's attitude toward the mouse and the sound of his words begin the poem on a gentle, humorous note.
The opening lines also introduces the poem's stanza form, meter, and rhyme scheme. The poem is written in six-line stanzas (sestets) that rhyme AAABAB. The A lines are written in iambic tetrameter (meaning they have four iambs—feet with an unstressed-stressed beat pattern—per line), while the B lines are written in iambic dimeter (meaning they have two iambs her line). This form is known as the habbie stanza or the Burns stanza (for more on this, see "Form").
There are variations on the iambic meter throughout the poem. Lines that, like lines 1-6, have feminine endings have an extra unstressed syllable at the end of the line. Some lines also begin with a stressed syllable. In line 1, the unusual stress on the first syllable calls extra attention to the word "wee," emphasizing the mouse's small size and the speaker's kindly attitude towards the small, helpless animal. The speaker's sympathy for the mouse will continue throughout the poem, but the tone will shift from the lightness and humor here to something more serious in the next stanza.